SRC TR

- 15

- 050

HPV2 TEST RULE DATA ADEQUACY REVIEW ( FINAL ): ACUTE TOXICITY TO FI SH AND ACUTE TOXICITY TO DAPHNIA

D

- gluco

- heptonic acid, monosodium salt, (2.xi.)

- , CASRN 31138

- 65

- 5

Prepared by:

Justine von Runnen, William Richards, Amy Nguyen, 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

Cynthia Bowie , Project Officer David Brooks, Work Assignment Manager Amy Benson, Alternate Work Assignment Manager

August 19 , 201 5Final HPV 2 Test Rule Data Adequacy Review (August 2015) CASRN 31138

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i TABLE OF CONTENTS

A. SOURCE MATERIAL USED FOR ASSESSMENT

................................ .............................. 1
B. EVALUATING DATA ADEQUACY

................................ ................................ ..................... 2
C. SUMMARY OF DATA ADEQUACY FOR TESTING REQUIRED BY TEST RULE

........ 2

D. DATA FOR SIDS ENDPOINTS

................................ ................................ .............................. 4

D

- 1. Acute Toxicity to Fish

................................ ................................ ................................ ....... 4

D

- 2. Acute Toxicity to Aquatic Invertebrates

................................ ................................ ............ 9
E. DATA ADEQUACY SUMMARY TABLE

................................ ................................ ........... 14Final HPV 2 Test Rule Data Adequacy Review (August 2015) CASRN 31138

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1 A. SOURCE MATERIAL USED FOR ASSESSMENT

This document evaluates two aquatic t oxicity test s for D

- gluco

- heptonic acid, monosodium salt, (2.xi.)

- (CASRN 31138

- 65

- 5) submitted by Harcros Chemicals, Inc. ( on May 28, 2015 and June 22, 2015 ) to EPA in response to the final test rule, “Testing of Certain High Production Volume Chemicals; Second Group of Chemicals” (76 FR 1067, J anuary 7, 2011; referred to herein as HPV2; available online at http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

2007

- 0531

- 0579 as of August 19 , 2015 ).

The HPV2 test rule requires testing of CASRN 31138

- 65

- 5 for the following toxicological endpoints: C1 ( aquatic toxicity, consisting of Test Group 1 [acute toxicity to fish, acute toxicity
to Daphnia , and toxicity to algae test s ] if log K ow < 4.2 or Test Group 2 [chronic toxicity to Daphnia and toxicity to algae test s ] if log K ow ≥ 4.2) , D (acute mammalian toxicity test), E1 (bacterial reverse mutation test), E2 (chromosomal aberration or micronucleus test), and F1 (combined repeated dose toxi city study with the reproduction/developmental toxicity screening test). Based on an estimated log K ow of

- 6.44 1 , Test Group 1 testing is required to determine the toxicity of CASRN 31138

- 65

- 5 to aquatic organisms.

Harc r os Chemicals, Inc. previously sub mitted acute toxicity to fish and acute toxicity to Daphnia studies t o address components of the C1 testing requirement promulgated in the final HPV2 test rule. These studies were reviewed by EPA 2 and determined to be inadequate to satisfy the acute toxic ity to fish and acute toxicity to Daphnia testing requirements primarily because the pH of the test solution preparations was adjusted to 7 prior to addition of the test organisms; this neutralization of the pH may have altered the toxicity of the test solutions to fish and Daphnia

. In July 2014, Harcros Chemicals

, Inc. submitted study plans to EPA for two aquatic toxicity
tests 3

, 4 with CASRN 31138

- 65

- 5. In response to EPA’s study plan review ( SRC Technical Report #14

- 030) , H arcros Chemicals

, Inc. submitted revised study plans to EPA o n October 13, 2014 5

. EPA reviewed the revised study plans and provided comments and recommendations for conducting adequate studies for the acute toxicity to fish and acute toxicity to Daphnia endpoints ( SRC Tec hnical Report #14

- 049)

. In May and June of 2015, H arc r os Chemicals, Inc. submitted the following studies to EPA to address components of the C1 testing requirement :

1 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 August 19, 2015.

2 Detailed reviews of previously submitted studies for the acute toxicity to fish and Daphnia endpoints are available online at http://www.regulations. gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0807 and http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0806 , respectively, as of August 19, 201 5.

3 Harris, S. 2014a. Study plan, Sodium glucoheptonate: Acute toxicity to rainbow trout. Test Facility: Harlan Laboratories Ltd. Study No. 41402585. Sponsor: Harcros Chemicals, Inc. Submitted to EPA in July 2014.

4 Harris, S. 2014b. Study plan, S odium glucoheptonate: Daphnia sp., 48

- hour acute immobilization test. Test Facility: Harlan Laboratories Ltd. Study No. 41402586. Sponsor: Harcros Chemicals, Inc. Submitted to EPA in July 2014.
5 Revised study plans for acute toxicity to fish and Daphn ia tests are available online at http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0825 as of August 19, 2015.Final HPV 2 Test Rule Data Adequacy Review (August 2015) CASRN 31138

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2 • Acute Toxicity to Rainbow Trout, submitted to EPA on June 22, 2015

• Acute Toxicity to Daphnia , submitted to EPA on May 28, 2015 (available online at http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0863 as of August 19 , 2015)

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

B. EVALUATING DATA ADEQUACY

Newly submitted d ata for the acute toxicity to fish and acute toxicity to Daphnia endpoint s 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 test rule requirements , study details were compared to current standard test guidelines to establish whether each study met current testing requirements. If the data available for the endpoint were determined to be inadequate, a basis for that co nclusion is supplied. A table summarizing data availability/adequacy for each SIDS endpoint is included in Section E of this document.

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

Evaluation of Data Submitted for the Acute Toxicity to Fish and Acute Toxicity to Daphnia Testing Requirements on May 28, 2015 and June 22, 2015: Data supplied in the May and June 2015 submissions are adequate to satisfy the acute toxicity to fish (component of C1) and acute toxicity to Daphnia (component of C1) testing requirements promulgated in the final HPV2 test rule.

Remaining HPV2 Test Rule Toxicity Testing Requirements: In several previous submissions (dated from December 18, 2012 to August 27, 2013), Harcros Chemicals, Inc. submitted data for toxicity to plants (algae) (component of C1), acute mammalian toxicity (D), gene mutation (E1), chromosomal aberrations (E2), and combined repeated

- dose toxicity with reproduction/developmenta l toxicity screening (F1 ) testing requirements promulgated in the final HPV2 test rule. These submissions included the following:

• Algal Inhibition Test, submitted for C1 testing requirement on April 23, 2013 (available online at http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0716 as of August 19 , 2015 )

• Acute Toxicity in the Rat

– Up and Down Procedure, submitted for D testing requirement on Ma rch 19, 2013 (available online at http://www.regulations.gov/#!documentDetail; D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0710 as of August 19 , 2015 )

• Reverse Mutation Assay “Ames test” using Salmonella typhimurium and Escherichia coli , submitted for E1 testing requirement on December 18, 2012 (available online atFinal HPV 2 Test Rule Data Adequacy Review (August 2015) CASRN 31138

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- 5

3 http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0688 as of August 19 , 2015 ) 1

• Micronucleus Test in the Mouse, submitted for E2 testing requirement on March 7, 2013 (available online at http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

2007

- 0531

- 0709 as of August 19 , 2015 )

• Oral (Gavage) Combined Repeat Dose Toxicity Study with Reproduction/Developmental Toxicity Screening Test in the Ra t (OECD 422), s ubmitted for F1 testing requirement on August 27, 2013 (available online at http://www.regulations.gov/#!documentDetail;D= EPA

- HQ

- OPPT

- 2007

- 0531

- 0790 as of August 19 , 2015 )

The data from the submissions above have been reviewed in separate document s (SRC Technical Report s #13

- 045 and #13

- 071 ). The data were determined to be adequate to satisfy the toxicity to plants (algae) (component of C1), acute mammalian toxicity (D), gene mutation (E1), chromosomal aberrations (E2) , and combined repeated

- dose toxicity with reproduction/developmental toxicity screening (F1 ) testing requirements

. Summary of Data Adequacy for Toxicological Endpoints: Adequate data have been submitted by Harcros Chemicals, Inc. for acute toxicity to fish, acute toxicity to Daphnia

, toxicity to plants (algae), acute mammalian toxicity, repeated

- dose toxicity, gene mutation, chromosomal aberration s, reproductive toxicity, and developmental toxicity endpoints.

There are no outstanding HPV2 test rule requirements for toxicity testing of CASRN 31138

- 65

- 5.

1 Harcros Chemical, Inc. submitted an amendment to this study report on May 22, 2013 (available online at http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0718 as of August 19, 2015) that indicated that formulated concentrations used in Experiments 1 and 2 were adjusted for water content (49.5%).Final HPV 2 Test Rule Data Adequacy Review (August 2015) CASRN 31138

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4 D. DATA FOR SIDS ENDPOINTS

D

- 1. Acute Toxicity to Fish

Summary of endpoint: A dequate

Study 1: 96

- Hour acute toxicity study in rainbow trout (Harris, 2015 a )

Title: Sodium g lucoheptonate CAS No. 31138

- 65

- 5: Acute t oxicity to r ainbow t rout

This study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: D

- gluco

- heptonic acid, monosodium salt, (2.xi.)

- (synonym: sodium glucoheptonate); CASRN 31138

- 65

- 5

Remarks: Amber

- colored liquid; B atch No. T

- 461; Purity: 39.94%; Composition: 25.78% Beta Sodium Glucoheptonate (CASRN 30090

- 50

- 3 1 ) 14.16% Alpha Sodium Glucoheptonate (CASRN 13007

- 85

- 7) 10.96% Polysaccharides or D egradation Products 0.3 ppm Cyanide 1113 ppm Ammonia 49.1% Water pH = 9.1 Specific Gravity = 1.293 % Solids = 50.9% No dextrose detected

Based on the compositional information provided, the purity of the test substance was 39.94 % a.i. 2 ( 25.78 % beta sodium glucoheptonate + 14.16 % alpha sodium glucoheptonate).

METHOD

Method/guideline followed: OECD TG 203 (1992; “Fish, Acute Toxicity Test”), Method C.1 of Commission Regulation (EC) No. 440/2008, and OPPTS 850.1075 ( 1996; “Fish Acute Toxicity Test, Freshwater and Marine”)

Test type: Acute F ish Toxicity Test (Semi

- Static)

GLP compliant?: Yes

1 30090

- 50

- 3 is not a valid CAS number according to the U.S. National Library of Medicine’s ChemIDplus website ( http://chem.sis.nlm.nih.gov/chemidplus/ ); however, based on the chemical name provided (beta sodium glucoheptonate), this component is assumed to be a stereoisomer of CASRN 31138

- 65

- 5.

2 Act ive ingredient (a.i.) = CASRN 31138

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- 5Final HPV 2 Test Rule Data Adequacy Review (August 2015) CASRN 31138

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5 Year study was performed: 2015

Test species: Rainbow trout ( Oncorhynchus mykiss ) obtained from Brow Well Fisheries Limited, lo cated in Hebden, near Skipton, Yorkshire, UK. Fish were acclimatized to test conditions for 7 days prior to exposure. The definitive test fish were juveniles that had a mean standard length of 3.9 cm and a mean weight of 0.72 g at the end of the definitive test. The loading rate was reported to be 0.25 g bodyweight/L.

Analytical monitoring: Yes

Exposure duration: 96 hours

Concentrations tested: Nominal : 0 and 100 mg a.i. /L (definitive and range

- finding tests)

Test method/conditions remarks:

Dilution W ater: Dechlorinated l aboratory tap water was partly softened to achieve a total hardness of approximately 140 mg CaCO 3 /L. At the start of the definitive test

, the dilution water h ad a particulate matter concentration of 0.1 mg/L, total water hardness of 1 34 mg CaCO 3 /L, alkalinity of 112.4 mg CaCO 3 /L, Total Organic Carbon (TOC) content of 2.0 mg C/L

, chemical oxygen demand of 1.36 mg O 2 /L

, conductivity of 297 μS/cm , and c hlorine content r anging f rom 0.00 8 to 0.0 3 mg Cl 2 /L.

Photoperiod: For both the range

- finding and definitive tests, the photoperiod was 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods.

Range

- Finding Test Procedure: In the range

- finding test, three fish w ere placed in a test vessel containing the test substance at a nominal concentration of 100 mg a.i./L and exposed for 96 hours under static conditions. A control group of three fish w as maintained under identical conditions but not exposed to the test substan ce. The rationale for selection of the nominal concentration of 100 mg a.i./L was based on data obtained from a Daphnia magna study (Harlan Study No. 41402586 ; reviewed under section D

- 2, “Acute Toxicity to Aquatic Invertebrates” of this report )

, which su ggested that toxicity to aquatic organisms would not be expected at this concentration

.

Test Conditions: Test vessels (25

- to 30

- L glass exposure vessel s ) contained 20 L of test solution and were covered to reduce evaporation. Test vessels were maintained at approximately 14°C in a temperature

- controlled room.

Test Solution Preparation: To prepare the 100 mg a.i./L test solution ( which was corrected for a purity of 39.94%), a nominal amount of test substance (5007.5 mg) was dissolved in dilution water and the volumeFinal HPV 2 Test Rule Data Adequacy Review (August 2015) CASRN 31138

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6 adjusted to 20 L. The solution was then stirred using a flat

- bladed mixer for approximately 1 minute to ensure homogeneity.

Analysis of Test Solutions: The stability and concentration of the test substance in the 100 mg a.i./L solution was verified by chemical analysis (HPLC

- MS) at 0 and 24 hours

. “The analytical methodology was not validated prior to the analysis of range

- finding samples; full validation was later conducted specifically at the concentration employed in the definitive test.” [Harris 2015 a , p. 33 ]

Definitive Test Procedure: Based on the re sults of the range

- finding test, a limit test was conducted at a concentration of 100 mg a.i./L. In the limit test, ten fish were placed in a test vessel containing the test substance at a nominal concentration of 100 mg a.i./L and exposed for 96 hours under semi

- static conditions with daily renewal. A control group of ten fish w as maintained under identical conditions but not exposed to the test substance.

Test Conditions: Test vessels (25

- to 30

- L glass exposure vessels ) contained 20 L of test solution and were covered to reduce evaporation. Test vessels received no auxiliary aeration and were maintained at approximately 14°C in a temperature

- controlled room.

Test Solution Preparation: To prepare the 100 mg a.i./L test solution (which was corrected for a purity of 39.94%), a nominal amount of test substan ce (5007.5 mg) was dissolved in dilution water and the volume adjusted to 20 L. The solution was then stirred using a flat

- bladed mixer for approximately 1 minute to ensure homogeneity.

Observations: Any mortalities and/or sub

- lethal effects were recor ded at 3, 6, 24, 48, 72

, and 96 hours after the start of exposure in the range

- finding and definitive tests

. The criteria for mortality included the absence of both respiratory movement and response to physical stimulation.

Physico

- Chemical Measurements : Wa ter temperature, pH
, and dissolved oxygen concentration s were measured and recorded daily throughout the test

. The temperature in the control replicate was also monitored hourly.

Analysis of Test Solutions: The stability and concentration of test substance in the 100 mg a.i./L solution was verified by chemical analysis (HPLC

- MS) at 0 and 72 hours (fresh media) and 24 and 96 hours (old media). Duplicate samples, as well as samples taken at 24 (fresh media), 48 (old and fresh m edia), and 72 (old media) hours

, were stored frozen for further analysis if necessary.Final HPV 2 Test Rule Data Adequacy Review (August 2015) CASRN 31138

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7 RESULTS

Endpoint value(s): Based on nominal concentrations , endpoint values from the definitive test

were: 96

- hour LC 50 > 100 mg a.i./L ; 96

- hour NOEC = 100 mg a.i./L ; 96

- hour LOEC > 100 mg a.i./L

Results remarks:

Range

- Finding Test Results: No mortalities or sub

- lethal effects were observed during the range

- finding test. Analytical measurements of the test substance in the 100 mg a.i./L test preparation at 0 and 24 hours ranged from 100 to 102 % of nominal concentrations. Therefore, the test substance was considered to be stable under test conditions.

Definitive Test Results: The test preparations were observed to be clear straw

- colored solutions throughout the test. No mortalities occurred during the definitive test. One of the 10 fish in the 100 mg a.i./L group was observed to be sitting on the bottom of the tank at the 6

- hour time point only . G iven that this effect was not observed at any other time point during the study, it was considered to be due to natural causes and was therefore not considered to be an adverse effect.

Physico

- Chemical Measurements: Throughout the test, pH ranged from 7.5 to 7.8 and dissolved oxygen ranged from 8.4 to 10.5 mg/L. At the start of the test, the temperature was 1 8°C ; however, the study author indicated that this was because the logger had been moved from storage at a higher temperature and had
not been able to acclimatize before this measurement was taken

. Temperature of media was maintained at 13

- 14 ° C for the remainder of the test.

Verification of Test Concentrations: Analytical measurements of the test substance in fresh test preparations at the nominal concentration of 100 mg a

. i

. /L were 77.6/89.4 mg a.i./L (duplicate samples) at 0 hours and 98.4 mg a.i./L at 72 hours

. Analytical measurements of the test substance in old (spent) test media at the nominal concentration of 100 mg a.i./L were 77.2 mg a.i./L at 24 hours and 94.5 mg a.i./L at 96 hours

. Regarding the value of 77.2 mg a.i./L observed in old test media at 24 hours, the study authors state: “ Given that preliminary work conducted, along with the range finding test showed that the test item was stable in the test system, this result was considered to be anomalous and was considered not to have had an effect on the outcome of the study. Consequently, the results are based on nominal test concentrations only ...” [Harris 2015 a , p. 15]

CONCLUSIONS

In the definitive test, t he 96

- hour LC 50 and LOEC f or rainbow trout w ere > 1 00 mg a.i./ L and the NOEC was 100 mg a.i./L, based on nominal concentrations.Final HPV 2 Test Rule Data Adequacy Review (August 2015) CASRN 31138

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8 STUDY RELIABILITY

[ 2 ] R eliable with restriction s

. The following deviations from test guidelines were noted :

(1) OECD TG 203 recommends that the temperature of the test media for Oncorhynchus mykiss be with in the range of 13

- 17 °C during the test; however, a temperature of 18°C was measured at the start of the definitive test. T he study author indicated that this was because the logger had been moved from storage at a higher temperature and had not been able to acclimatize before this measurement was taken

. T emperature of the test media was maintained at 13

- 14°C for the remainder of the test.

(2) OECD TG 203 states that if measured concentrations of the test substance (i.e., the active ingredient) deviate by more than 20% from the nominal concentrations throughout the test, results should be based on measured (rather than nominal) concentrations. In the definitive test, m easured concentrations in one duplicate sample of the 0

- hour (fresh) sample and in the 24

- hour (old) sample were lower than expected (7 7.6 % and 77 .2 % of nominal concentrations , respectively). However, an alysis of the duplicate 0

- hour (fresh) sample showed a measured test substance concentration of 89 .4 % of the nominal concentration

. Analysis of the duplicate 24

- hour sample was not possible as it was accidentally discarded

. 1 The low measured concentration in the 24

- hour (old) sample might be anomalous given that (1) measured concentrations in the 72

- hour (fresh) and 96

- hour (old) samples ranged from 9 4. 5

- 98 .4 % of nominal and (2) measured concentrations in the range

- finding t est ranged from 100

- 102%

, suggesting that the test substance is stable.

REFERENCE

Harris, S. 20 15 a

. Sodium g lucoheptonate CAS No. 31138

- 65

- 5 : Acute toxicity to rainbow trout. Harlan Laboratories Ltd., Shardlow, Derbyshire, UK. Study No. 4 1402585 . Sponsor: Harcros Chemicals, Inc., Dalton, GA.

Text in quotes is taken directly from Harris (201 5 a ).

1 Although an explanation was not provided in the study report, Harcros Chemicals, Inc. notified EPA in an email dated April 20, 2015 (addressed from Ms. Ann Marie Savini of Harcros Chemicals, Inc. to Mr. John Schae ffer of EPA) that the duplicate 24

- hour sample was discarded in error and could not be analyzed.Final HPV 2 Test Rule Data Adequacy Review (August 2015) CASRN 31138

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9 D

- 2 . Acute Toxicity to Aquatic Invertebrates

Summary of endpoint: A dequate

Study 1: 48

- Hour a cute toxicity study in Daphnia magna (Harris , 201 5b )

Title: Sodium g lucoheptonate CAS No. 31138

- 65

- 5 : Daphnia sp., 48

- Hour a cute i mmobilization t est

This study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: D

- gluco

- heptonic acid, monosodium salt, (2.xi.)

- (synonym: sodium glucoheptonate); CASRN 31138

- 65

- 5

Remarks: Amber

- colored liquid; Batch No. T

- 461; Purity: 39.94%; Composition: 25.78% Beta Sodium Glucoheptonate (CASRN 30090

- 50

- 3 1 ) 14.16% Alpha Sodium Glucoheptonate (CASRN 13007

- 85

- 7) 10.96% Polysaccharides or Degradation Products 0.3 ppm Cyanide 1113 ppm Ammonia 49.1% Water pH = 9.1 Specific Gravity = 1.293 % Solids = 50.9% No dextrose detected

Based on the compositional information provided, the purity of the test substance was 39.94% a.i. 2 (25.78% beta so dium glucoheptonate + 14.16% alpha sodium glucoheptonate).

METHOD

Method/guideline followed: OECD TG 202 (2004; “ Daphnia sp., Acute Immobilisation Test”, M ethod C.2 of Commission Regulation (EC) No. 440/2008

, and OPPTS 850.1010 ( 1996; “Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids”).

Test type: Acute Daphnid Toxicity Test (Static)

GLP compliant?: Yes

1 30090

- 50

- 3 is not a valid CAS number according to the U.S. National Library of Medicine’s ChemIDplus website ( http://chem.sis.nlm.nih.gov/chemidplus/ ); however, based on the chemical name provided (beta sodium glucoheptonate), this component is assumed to be a stereoisomer of CASRN 31138

- 65

- 5.

2 Active ingredient (a.i.) = CASRN 31138

- 65

- 5Final HPV 2 Test Rule Data Adequacy Review (August 2015) CASRN 31138

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- 5

10 Year study was performed: 2015

Test species: Water flea ( Daphnia magna ); < 24 hours old; obtained from in

- house laboratory cultures

Analytical monitoring: Yes

Exposure duration: 48 hours

Concentrations tested: Nominal: 0, 0.10, 1.0, 10, and 100 mg a.i./L (range

- finding test); 0 and 100 mg a.i./L (definitive test)

Test method/conditions remarks:

Dilution Water: Reconstituted water (ISO medium) was used for both t he range

- finding and definitive tests. The reconstituted water had an approximate theoretical total hardness of 250 mg CaCO 3 /L. T he reconstituted water was diluted 50:50 with deionized reverse osmosis w ater to achieve a total water hardness that complied with OPPTS

- recommended water characteristics. At the start of the definitive test, the dilution water had a total water hardness of 146 mg CaC O 3 /L and an alkalinity of 27.6 mg CaC O 3 /L

. Photoperiod: For both the range

- finding and definitive tests, the photoperiod was 16 hours light (intensity: not indicated for range

- finding study; 565 to 626 lux for definitive study) and 8 hours darkness with 20 minute dawn and dusk transition periods.

Range

- Finding Test Procedure: In the range

- finding test, groups of ten D. magna per vessel (one vessel per concentration) were exposed to the test substance at nominal concentration s of 0.10, 1.0, 10, and 100 mg a.i./L for 48 hours under static conditions. A control group of ten D. magna w as maintained under identical conditions but not exposed to the test substance. All test concentrations were corrected for a test substance purity of 39.94%.

Test Conditions: Test vessels (250

- mL glass jars ) contained 20 0 mL of test media and were covered to reduce evaporation. Test vessels were maintained in a temperature

- controlled room at 21 °C.

Test Solution Preparation: A nominal amount of test substance ( 250.4 mg) was dissolved in dilution w ater and the volume adjusted to 1 L to give the 100 mg a.i./L test concentration (corrected for a purity of 39.94%) from which a series of dilutions was made to obtain the remaining nominal concentrations of 0.10, 1.0, and 10 mg a.i./L

. Each prepared test solution was then inverted several times to ensure adequate mixing and homogeneity.Final HPV 2 Test Rule Data Adequacy Review (August 2015) CASRN 31138

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11 Analysis of Test Solutions: The stability and concentration of test substance in the 100 mg a.i./L solution was verified by chemical analysis (HPLC

- MS) at 0 and 48 hours. “The analytical methodolo gy was not validated prior to the analysis of range

- finding samples; full validation was later conducted specifically at the test concentration employed in the definitive test.” [Harris 2015 b , p. 32]

Definitive Test Procedure: Based on the results of the range

- finding test, a limit test was conducted at a concentration of 100 mg a.i./L. In the limit test, four replicates of five D. magna were exposed to the test substance at a nominal concentration of 100 mg a.i./L (corrected for a purity of 39.94%) for 48 hours under static conditions. Additionally, four control replicates of five D. magna were maintained under identical conditions but not exposed to the test substance.

Test Conditions: Test vessels (250

- mL gla ss jars ) contained 2 00 mL o f test solution and were covered to reduce evaporation. Test vessels were not aerated and were maintained in a temperature

- controlled

room at 21°C

. Test Solution Preparation: To prepare the 100 mg a.i./L test solution (corrected for a purity of 39.94%), a nominal amount of test substance ( 250.4 mg) was dissolved in dilution wa ter and the volume adjusted to 1 L. The prepared test solution was then inverted several times to ensure adequate mixing and homogeneity.

Analysis of Test Solutions: Samples from the control and the 100 mg a.i./L test groups were taken for quantitative analysis (HPLC

- MS) at 0 and 48 hours. Samples taken at 0 hours were from t he bulk test preparation and samples taken at 48 hours were from th e pooled repl i cates. The 48

- hour samples were stored frozen prior to analysis ; however, the 0

- hour samples were not stored frozen for the initial 2 days after sampling due to a technical error . This was not considered to have had an impact on the
outco me of the test given that the achieved measured concentrations were close to nominal concentrations . Additionally, d uplicate samples were taken and stored frozen for further analysis if necessary.

Observations: Any immobilization or adverse effects were recorded at 24 and 48 hours in the range

- finding and definitive tests

. Daphnia were considered to be immobilized if they were unable to swim for approximately 15 seconds after gentle agitation.

Physico

- Chemical Measurements : Water temperature was measu red and recorded daily through out the test and the temperature was also measured every hour in one control replicate. Dissolved oxygen concentrations and pH

w ere measured and recorded at 0 and 48 hours.Final HPV 2 Test Rule Data Adequacy Review (August 2015) CASRN 31138

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12 RESULTS

Endpoint value(s): Based on nominal concentrations, endpoint values from the definitive test were: 48

- hour EC 50 > 100 mg a.i./L; 48

- hour NOEC = 100 mg a.i./L ; 48

- hour LOEC > 100 mg a.i./L

Results remarks:

Range

- Finding Test Results: The measured test substance concentration in the 100 mg a.i./L group was 93% of the nominal concentration at 0 hours and 87% of the nominal concentration at 48 hours. Therefore, the test substance was considered to be stable under test conditions. No imm obilization was observed in the 0.10 or 1.0 mg a.i./L test groups. Immobilization was noted in a single daphni d each from the 10 and the 100 mg a.i./L test groups at 48 hours ; however, this was not considered to be an adverse effect as no more than 10% im mobilization occurred. Based on these findings, a single limit concentration of 100 mg a.i./L was sel ected for the definitive test.

Definitive Test Results: The test preparations were observed to be clear and colorless solutions throughout the test. No immobilization or abnormal signs were o bserved d uring the definitive test.

Physico

- Chemical Measurements: Throughout the test, temperature was maintained at 21 ° C, pH ranged from 7.5 to 8.3 , and dissolved oxygen ranged from 8.4 to 9.1 mg/L.

Verification of Test Concentrations: Analysis of the 100 mg a

. i

. /L test solutions at 0 and 48 hours showed measured test concentrations to range from 80 to 86% of nominal concentrations ; therefore, the results were based on nominal concentrations.

CONCLUSIONS

In the definitive test, the 48

- hour EC 50 and L OEC for Daphnia magna were > 100 mg a.i./L and the NOEC was 100 mg a.i./L, based on nominal concentrations.

STUDY RELIABILITY

[2] Reliable with restriction s . The following deviation from test guidelines w as noted: According to Method C.2 of Commission Regulation (EC) No. 440/2008, t he alkalinity of the dilution water should be 0.8 mmol/L ; however, as noted by the study author, the alkalinity of the dilution water used in t his test was 27.6 mg CaC O 3 /L (equivalent to 0.27576 mmol/L), which is less than the guideline

- recommended value

.Final HPV 2 Test Rule Data Adequacy Review (August 2015) CASRN 31138

- 65

- 5

13 REFERENCE

Harris, S. 201 5 b

. Sodium glucoheptonate CAS No. 31138

- 65

- 5 : Daphnia sp., 48

- h our acute i mmobilization test. Harlan Laboratories Ltd., Shardlow, Derbyshire, UK. Study No. 41 402586. S ponsor: Harcros Chemicals, Inc., Dalton, GA.

Text in quotes is taken directly from Harris (2015 b ).Final HPV 2 Test Rule Data Adequacy Review (August 2015) CASRN 31138

- 65

- 5

14 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 a Yes Yes No Health Effects Acute Toxicity a Yes Yes No Repeated

- Dose Toxicity a Yes Yes No Gene Mutation a Yes Yes No Chromosomal Aberrations a Yes Yes No

Reproductive Toxicity a Yes Yes No Developmental Toxicity a Yes Yes No

a Data were reviewed in a separate report.