SRC TR

- 13

- 059

HPV 2 TEST RULE DATA ADEQUACY REVIEW ( REVISED FINAL ):

ACUTE TOXICITY TO DAPHNIA

D

- gluco

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

- , CASRN 31138

- 65

- 5

Prepared by:

Amy Nguyen, Lisa Ingerman, Jennifer Rayner, Teresa Manyin, and William Richards

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

January 14 , 2014Final HPV 2 Chemical Data Adequacy Review CAS RN 31138

- 6 5

- 5

1 A. SOURCE MATERIAL USED FOR ASSESSMENT

This document evaluates a toxicity test for D

- gluco

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

- (CASRN 31138

- 65

- 5) submitted by Harcros Chemicals, Inc. ( on June 12 , 2013 ) 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 January 14, 2014 ).

T he HPV2 test rule requires testing of CASRN 31138

- 65

- 5 for the following toxic ological endpoints: C1 (consisting of acute toxicity to fish, acute toxicity to Daphnia , and toxicity to algae testing if log K ow < 4.2 or chronic toxicity to Daphnia and toxicity to algae testing 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 toxicity study with the reproduction/developmental toxicity screening test). Since the log K ow of CASRN 31138

- 65

- 5 is estimated to be

- 6.44 1 , which is < 4.2, the C1 aquatic toxicity testing requirement is considered to include acute toxicity to fish, acute toxicity to Daphnia , and toxicity to algae testing.

The June 12, 2013 submission from Harcros Chemicals, Inc. included the following:

 Dap hnia sp., 48

- hour A cute I mmobilization T est , submitted for the C1 testing requirement ( study report available online at http://www.regulations.gov/ #!documentDetail ;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0719 as of January 14, 2014 )

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

B. EVALUATING DATA ADEQUACY

Data available for the daphnid SIDS toxicity endpoint 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. Studies for a common endpoint are grouped together in the document and an endpo int adequacy assessment is provided near the top of the first page of the summaries for that endpoint. If the data available for the endpoint were determined to be inadequate, a basis for that conclusion is supplied. A table summarizing data availability /adequacy for each SIDS endpoint is included in Section E of this document.

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 January 14, 2014

.Final HPV 2 Chemical Data Adequacy Review CAS RN 31138

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

Data supplied in the June 12, 2013 submission are not considered adequate to satisfy the acute toxicity to Daphnia ( component of C 1) test ing requirement promulgated in the final HPV2 test rule. This study is not adequate for the following reasons: (1) The pH of the test solution preparations in the definitive test (and in the range

- finding and post

- study tests) was adjust ed to 7 prior to the addition of test organisms. If the test item causes a change in pH that increases the toxicity of the solution, then the toxicity is considered to be the result of the test item. Given that the pH of the test substance is 9.62, the a ddition of the test substance to the dilution water (consisting of a 50:50 mixture of reconstituted water [pH of 7.8 ± 0.2] and deionized reverse osmosis water [pH not provided]) would be expected to increase the pH. If the resulting pH is outside of the physiologically acceptable range for Daphnia of approximately 6 to 9 (per OECD TG 202), then toxicity may result from the increase in pH caused by the addition of the test substance. Any toxicity resulting from a change in pH caused by the test substance is considered to be a toxic effect of the test substance. Therefore, by adjusting the pH of test solutions to 7 before exposing the Daphnia to these solutions, the testing laboratory may have altered the toxicity of the test item to Daphnia . The study re sults are not representative of the toxicity of the unaltered substance. (2) Substantially different findings in the range

- finding and definitive tests raise concerns over the reliability of the test methods. In the range

- finding test, 50% immobilization (5/10 daphnids) was observed at 24 hours and 100% immobilization (10/10 daphnids) was obser ved at 48 hours at the maximum concentration tested (872 mg a.i./L). In contrast, no effects were observed at 48 hours in the definitive test (maximum test concentration of 889 mg a.i./L) or in the post

- study test (maximum test concentration of 872 mg a.i ./L). As presented in the report, similar test procedures were used in the range

finding, definitive, and post

- study tests (all included adjustment of test solutions to pH 7 prior to the introduction of the daphnids). The investigators conducted a post

- s tudy test which confirmed the results of the definitive test, but do not provide a possible explanation for the dissimilar results between the range

- finding and definitive/post

- study
tests.

The acute toxicity to Daphnia test needs to be repeated without a djustment of pH in the prepared test solutions.

In five previous submissions ( dated from December 18, 2012 to April 24, 2013), Harcros Chemicals, Inc. submitted data for acute toxicity to fish (component of C1), toxicity to plants (algae) (component of C1 ), acute mammalian toxicity (D), gene mutation (E1), and chromosomal aberrations (E2) testing requirements. These submissions included the following:

 Acute Toxicity to Rainbow Trout, submitted for C1 testing requirement on April 24, 2013 (available online at http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

OP PT

- 2007

- 0531

- 0715 as of January 14, 2014 )

 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 January 14, 2014 )Final HPV 2 Chemical Data Adequacy Review CAS RN 31138

- 6 5

- 5

3  Acute Toxicity in the Rat

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

- HQ

- OPPT

- 2007

- 0531

- 0710 as of January 14, 2014 )

 Reverse Mutation Assay “Ames test” using Salmonella typhimurium and Escherichia coli , submitted for E1 testing requirement on December 18, 2012 (available online at http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2007

- 053 1

- 0688 as of January 14, 2014 ) 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 January 14, 2014 )

The data from the submissions above have been reviewed in a separate document (SRC Technical Report # 1 3

- 045 ). The data were determined to be adequate to satisfy the toxicity to plants (algae) ( component of C 1) , acute mammalian toxicity (D), gene mutation (E1)

, and chromosomal aberrations (E2) test ing requirement s, but not adequate to satisfy the acute toxicity to fish (component of C 1) testing requirement

.
On August 27, 2013, Harcros Chemicals, Inc. submitted the following report for the combined repeated

- dose toxicity study with the reproduction/developmental toxicity screening test (F1) requirement:

 Oral (Gavage) Combined Repeat Do se Toxicity Study with Reproduction/Developmental Toxicity Screening Test in the Rat (OECD 422), submitted via email from A.M. Savini (Harcros Chemicals, Inc.) to J. Schaeffer (EPA) on August 27, 2013

This study was reviewed in a separate report (SRC Tech nical Report #13

- 071) and was determined to be adequate to satisfy the combined repeated

- dose toxicity with reproduction/developmental toxicity screening (F1 ) testing requirement promulgated in the final HPV2 test rule

.
Summary of Data Adequacy for Toxico logical Endpoints: Data submitted by Harcros Chemicals, Inc. for the acute toxicity to fish and acute toxicity to Daphnia endpoints are not considered adequate; these tests need to be repeated without pH adjustment of test solutions. Adequate data have been submitted by Harcros Chemicals, Inc. for toxicity to plants (algae), acute mammalian toxicity, repeated

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

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 January 14, 2014 ) that indicated that formulated concentrations used in Experiments 1 and 2 were adjusted for water content (49.5%).Final HPV 2 Chemical Data Adequacy Review CAS RN 31138

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- 5

4 D . DATA FOR SIDS ENDPOINTS

D

- 1 . Acute Toxicity to Aquatic Invertebrates

Summary of endpoint: Not a dequate

Study 1 : Acute toxicity study in Daphnia magna ( Harris , 201 3 )

Title: Sodium Glucoheptonate: Daphnia sp., 48

- Hour Acute Immobilization Test

This study is: Adequate Not Adequate

This study is not adequate for the following reasons: (1) The pH of the test solution preparations in the definitive test (and in the range

- finding and post

- study tests) was adjusted to 7 prior to the addition of test organisms. If the test item causes a change in pH that increases the toxicity of the solution, then the toxicity is considered to be the result of the test item. Given that the pH of the test substance is 9.62, the addition of the test substance to the dilution water (consisting of a 50:50 mixture of reconstituted water [pH of 7.8 ± 0.2] and deioni zed reverse osmosis water [pH not provided]) would be expected to increase the pH. If the resulting pH is outside of the physiologically acceptable range for Daphnia of approximately 6 to 9 (per OECD TG 202), then toxicity may result from the increase in pH caused by the addition of the test substance. Any toxicity resulting from a change in pH caused by the test substance is considered to be a toxic effect of the test substance. Therefore, by adjusting the pH of test solutions to 7 before exposing the D aphnia to these solutions, the testing laboratory may have altered the toxicity of the test item to Daphnia . The study results are not representative of the toxicity of the unaltered substance. (2) Substantially different findings in the range

- finding and def initive tests raise concerns over the reliability of the test methods. In the range

- finding test, 50% immobilization (5/10 daphnids) was observed at 24 hours and 100% immobilization (10/10 daphnids) was observed at 48 hours at the maximum concentration te sted (872 mg a.i./L). In contrast, no effects were observed at 48 hours in the definitive test (maximum test concentration of 889 mg a.i./L) or in the post

- study test (maximum test concentration of 872 mg a.i./L). As presented in the report, similar test procedures were used in the range

finding, definitive, and post

- study tests (all included adjustment of test solutions to pH 7 prior to the introduction of the daphnids). The investigators conducted a post

- study test which confirmed the results of the de finitive test, but do not provide a possible explanation for the dissimilar results between the range

- finding and definitive/post

- study
tests.

TEST SUBSTANCE

Identity: D

- gluco

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

- (synonym: sodium glucoheptonate); CASRN 31138

- 65

- 5Final HPV 2 Chemical Data Adequacy Review CAS RN 31138

- 6 5

- 5

5 Remarks: Dark brown liquid; Batch No. 921000100; purity reported as 49.5% (with 50.5% water) in study report; however, in emails from A.M. Savini (Harcros Chemicals, Inc.) to J. Schaeffer (EPA) on March 5, 2013 and May 14, 2013, the com position of the test substance was reported as follows: 23.4% Beta Sodium Glucoheptonate (CASRN 30090

- 50

- 3 1 ) 20.62% Alpha Sodium Glucoheptonate (CASRN 13007

- 85

- 7) 6.88% Polysaccharides or Degradation Products 2.84 ppm Cyanide 1038 ppm Ammonia 49.1% Water pH = 9.62 Specific Gravity = 1.28 % Solids = 50.9% No dextrose detected The above composition is identical to that provided by Harcros Chemicals, Inc. in its proposal for testing, available at online at http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

OPPT

- 2007

- 0531

- 0639 as of January 14, 2014 . Based on this information, the purity of the test substance was 44.0 2% a.i. 2 (23.4% beta sodium glucoheptonate + 20.62% alpha sodium glucoheptonate).

METHOD

Method/guideline followed: OECD TG 202 ( 2004; “ Daphnia sp., Acute Immobili s ation Test”

, referenced as method C.2 of Commission Regulation (EC) No. 440/2008 ) and OPPTS 850.1010 (“Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids”)

.
Test type: Acute Daphnid Toxicity Test ( Static )

GLP compliant?: Yes

Year study was performed: 201 3

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

- house cultures

Analytical monitoring: Yes , only at selected test concentrations (in the definitive test, only the highest concentration was measured analytically)

Exposure duration: 48 hours

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 Chemical Data Adequacy Review CAS RN 31138

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- 5

6 Concentrations tested: Nominal concentrations (definitive test) : 0, 202, 364, 647, 1131, and 2020 mg test item/L; Nominal c oncentrations adjusted for 44.02% purity (definitive test) : 0, 8 8. 9, 160, 284, 498, and 889 mg a.i./L. 1

Test method/conditions remarks:

Test water: Reconstituted water was prepared using d eionized water with a conductivity of < 5 μS/cm. The reconstituted water was adjusted to a pH of 7.8 ± 0.2. Reconstituted water was diluted in deionized water to obtain a theoretical total hardness of approximately 125 mg/L. The resulti ng water was used in both the range

- finding and definitive tests.

“At the start of the definitive test the diluent had a total water hardness of 148 mg/L as CaCO 3 and an alkalinity of 26 mg/L as CaCO 3
. ” [Harris 2013, p. 11]

Range

- Finding Test Procedure: In the range

- finding test, daphnids were exposed to a series of nominal test concentrations of 0, 0.08 7 2 , 0.8 7 2

, 8

. 7 2

, 87. 2 , and 8 7 2 mg a.i./L 2 for 48 hours.

To prepare the stock solution ( 87 2 mg a.i./L ) , 1980 mg of test item was dissolved in reconstituted water and the volume adjusted to 1 liter . A series of dilutions was made to prepare test solutions of 0.08 7 2

, 0.8 7 2 , 8. 7 2

, and 8 7

. 2 mg a.i./L

. “Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity, and the pH of each was adjusted to pH 7.” [Harris 2013, p. 11]

“In the range

- finding test 10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room at 21 oC with a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transit ion periods. [Only one test vessel was used per test concentration.] Each 250 mL test and control vessel contained 200 mL of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilized Daphnia magna were recorded. ” [Harris 2013, p. 11]

“A sample of each test concentration was taken for chemical analysis at 0 and 48 hours in order to determine the stability of the test item under test conditions. All samples were stored at approximately

- 20 °C prior to analysis. Only concentrations with in the range to be used for the definitive test [ 87. 2 and 87 2 mg a.i./L ] were analyzed. ” [Harris 2013, p. 1 1 ]

1 The study author erroneously reported nominal concentrations of 100, 180, 320, 560 , and 1000 mg a.i./L

, respectively, based on an incorrect purity of 49.5%

. The correct test substance purity w as 44.02%. The concentrations presented in the text above have been corrected for 44.02% purity.
2 The study author reported n ominal concentrations of 0, 0.10, 1.0, 10, 100, and 1 000 mg a.i./L , which were calculated by adjusting for a purity of 50.5% (water content of 49.5%). (The study author noted that a purity of 50.5% was erroneously used during the range

- finding study; the laboratory had been informed that the test substance purity was 49.5%

. ) The correct test substance purity was 44.02%. The concentrations presented in the text above have been corrected for 44.02% purity.Final HPV 2 Chemical Data Adequacy Review CAS RN 31138

- 6 5

- 5

7 Analysis of test substance concentrations in test solutions was conducted using HPLC

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

- finding samples; full validation was later conducted specifically over the test concentration range employed in the definitive test.” [Harris 2013, p. 31 ]

Definitive Test Proced ure: Based on the results of the range

- finding test, the concentrations used in the definitive test were 0, 8 8. 9, 160, 284, 498, and 889 mg a.i./L 1

.

“A n amount of test item (10,100 mg) was dissolved in reconstituted water and the volume adjusted to 5 liters [to prepare the test concentration of 889 mg a.i./L]. Aliquots (100, 180, 320 and 560 mL) of the [889 mg a. i

. /L ] test concentration w ere added to a final volume of 1 liter to give the remaining test concentrations

... ” [Harris 2013, p. 12]

“Each stock prepared concentration was inverted several times to ensure adequate mixing and homogeneity and the pH of each was adjusted to pH 7.” [Harris 2013, p. 12]

“In the definitive test 250 mL glass jars containing 200 mL of test preparation were used. At the start of the test 5 daphnids were placed in each test and control vessel at random, in the test preparations. Four replicate test and control vessels were p repared. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room at 20 °C to 21 °C with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods with a light inte nsity ranging from 645 to 682 lux. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated. ” [Harris 2013, p. 12]

“ The test preparations were not renewed during the exposure period. Any immobilization or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia were considered to be immobilized if they were unable to swim for approximately 15 seconds afte r gentle agitation. ” [Harris 2013, p. 12]

“Water temperature and light intensity were recorded daily throughout the test. Dissolved oxygen concentrations and pH were recorded at the start and termination of the test. ” [Harris 2013, p. 12]

For the defini tive test, “ Water samples were taken from the control and each test group (replicates R 1 - R 4 , pooled) at 0 and 48 hours for quantitative analysis. Samples were stored at approximately

- 20 °C prior to analysis. ” [Harris 2013, p. 13]

Only samples at the NOEC ( i.e., 889 mg a.i./L ) were analyzed. Analysis of test substance concentrations in test solutions was conducted using HPLC

- MS.

1 The study author erroneously reported nominal concentrations of 100, 180, 320, 560 , and 1000 mg a.i./L

, respectively, based on an incorrect purity of 49.5%

. The correct test substance purity w as 44.02%. The concentrations presented in the text above have been corrected for 44.02% purity.Final HPV 2 Chemical Data Adequacy Review CAS RN 31138

- 6 5

- 5

8 Post

- Study Test Procedure :

“ Whilst significant immobilization was observed to have occurred at [the highest test concentration] in the range fin ding test, no immobilization occurred at any test concentration employed in the definitive test. It was therefore considered appropriate to conduct a post

- study experiment in order to confirm the results obtained. ” [Harris 2 013, p. 13]

In the post

- study experiment, Daphnia magna were exposed to nominal test concentrations of 0, 0.08 72 , 0.8 72 , 8. 72

, 87.2 and 872 mg a

. i

. /L 1

.

“ An amount of test item (1980 mg) was dissolved in reconstituted water and the volume adjusted to 1 liter to give the [8 72 mg a

. i

. /L ] test concentration. Serial dilutions were made in reconstituted water to give the remainder of the test concentrations. ” [Harris 2013, p. 13]

“ Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity, and the pH of each was adjusted to pH 7. ” [Harris 2013, p. 13]

“ In the post

- study experiment 10 daphnids were placed in each test an d control vessel and maintained in a temperature controlled room at 21 °C with a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. [Only one test vessel was used per test concentration.] Each 250 mL test and control vessel contained 250 mL of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilized Daphnia magna were recorded. ” [Harris 2013, p. 13]

“ A sample of each test concentration was taken for chemical analysis at 0 and 48 hours in order to determine the stability of the test item under test conditions. All samples were stored at approximately

- 20 °C prior to analysis. ” [Harris 20 13, p. 1 4 ] Only samples at the NOEC ( i.e., 8 72 mg a.i./L ) w ere analyzed

.

RESULTS

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

- hour E C 50 > 889 mg a.i./L ; 48

- hour LOEC ( immobility ) > 889 mg a.i ./L; 48

- hour NOEC ( immobility ) = 889 mg a.i./L

Results remarks: Range

- Finding Test Results: No immo bilization was observed at 0, 0.08 7 2

, 8

. 72

, or 87.2 mg a.i./L. At 0.872 mg a.i./L, 10% of the daphnids (1/10) were immobilized at 48 hours; this observation was most likely unrelated to treatment, since no immobility was observed at 8

. 72 or 87.2 mg a.i./L

. At the highest test concentration (8 72 mg a.i./L) , 50% of the daphnids (5/10) were immobilized at 24 hours; 100% (10/10) were immobilized at 48 hours.

1 The study author erroneously reported nominal concentrations of 0.10, 1.0, 10, 100 , and 1000 mg a.i./L

, respectively, based on an incorrect purity of 50 .5%

. The correct test substance purity w as 44.02%. The concentrations presented in the text above have been corrected for 44.02% purity.Final HPV 2 Chemical Data Adequacy Review CAS RN 31138

- 6 5

- 5

9 Analytical measurements of the test substance in the 8 7.2 and 872 mg a.i./L test preparations at 0 and 48 hours ranged from 83% to 94 % of nominal concentrations for the range

- finding test

.

Definitive Test Results: No immo bilization was observed in the definitive test at nominal concentrations up to 889 mg a.i./L.

Throughout the test, the test preparations rang ed from an extremely pale amber

- colored solution (seen at 88.9 mg a.i./L ) t o a dark amber solution (seen at 889 mg a.i./L )

.
Daily water temperature measurements, measured every 24 hours at all test concentrations, ranged from 20 to 21 ° C

. “The water temperature which was also recorded in the control vessel approximately every hour us ing a Testo temperature logger... ranged from 20 ° C to 21 ° C.” [Harris 2013, p. 16] The pH (measured at 0 and 48 hours) was 6. 8

- 7. 5. Dissolved oxygen (measured at 0 and 48 hours) was 7.5

- 9.0 mg/L (8 4

- 100 % saturation).

“Analysis of the test preparations [at 889 mg a.i./L] at 0 and 48 hours [from the definitive test]

... showed measured test concentrations to range from 80% to 83% of nominal value and so it was considered justifiable to estimate EC 50 values in terms of the nominal test concentrations only

. ” [Harris 2013, p

. 16]

Post

- Study Test Results: No immobilization was observed in the post

- study test a t nominal concentrations up to 872 mg a.i./L.

“ Chemical a nalysis of the [ 872 mg a.i./L] test preparation at 0 and 48 hours [from the post

- study test]

... showed [that] near nominal concentrations were obtained [range of 88% to 92% of nominal concentrations] indicating that the test system was correctly dosed. It was therefore considered [by the study author] that the immobilization observed in the range finding test was due to contamination rather than a true toxic effect. ” [Harris 2013, p. 16]

CONCLUSIONS

In the definitive test, t he 48

- hour EC 50 and NOEC for Daphnia magna were determined to be

> 889 m g a.i. /L and 889 m g a.i./L, respectively, based on nominal concentrations.

STUDY RELIABILITY

[ 3 ] Not r eliable. This study is not reliable for the following reasons: (1) The pH of the test solution preparations in the definitive test (and in the range

- finding and post

- study tests) was adjusted to 7 prior to the addition of test organisms. If the test item causes a change in pH that increases the toxicity of the solution, then the toxicity is considered to be the result of the test item. Given that the pH of the test substance is 9 .62, the addition of the test substance to the dilution water (consisting of a 50:50 mixture of reconstituted water [pH of 7.8 ± 0.2] and deionized reverse osmosis water [pH not provided]) would be expected to increase the pH. If the resulting pH is outsi de of theFinal HPV 2 Chemical Data Adequacy Review CAS RN 31138

- 6 5

- 5

10 physiologically acceptable range for Daphnia of approximately 6 to 9 (per OECD TG 202), then toxicity may result from the increase in pH caused by the addition of the test substance. Any toxicity resulting from a change in pH caused by the test substance is considered to be a toxic effect of the test substance. Therefore, by adjusting the pH of test solutions to 7 before exposing the Daphnia to these solutions, the testing laboratory may have altered the toxicity of the test item to Daphnia

. The study results are not representative of the toxicity of the unaltered substance. (2) Substantially different findings in the range

- finding and definitive tests raise concerns over the reliabilit y of the test methods. In the range

- finding test, 50% immobilization (5/10 daphnids) was observed at 24 hours and 100% immobilization (10/10 daphnids) was observed at 48 hours at the maximum concentration tested (872 mg a.i./L). In contrast, no effects w ere observed at 48 hours in the definitive test (maximum test concentration of 889 mg a.i./L) or in the post

- study test (maximum test concentration of 872 mg a.i./L). As presented in the report, similar test procedures were used in the range

finding, defi nitive, and post

- study tests (all included adjustment of test solutions to pH 7 prior to the introduction of the daphnids) . The investigators conducted a post

- study test which confirmed the results of the definitive test, but do not provide a possible
exp lanation for the dissimilar results between the range

- finding and definitive /post

- study
tests.

REFERENCE

Harris

, S . (201 3) Sodium glucoheptonate: Daphnia sp., 48

- hour acute immobilization test. Performing Laboratory: Harlan Laboratories Ltd., Shardlow, Derbyshire, UK. Study No. 41104695. Sponsor: Harcros Chemicals, Inc., Dalton, GA.

Text in quotes is taken directly from Harris (2013).Final HPV 2 Chemical Data Adequacy Review CAS RN 31138

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- 5

11 E

. DATA ADEQUACY SUMMARY TABLE

Endpoint Study Information Available Acceptable Data Gap

Aquatic Effects Acute Toxicity to Fish a Yes No Yes Acute Toxicity to Aquatic Invertebrates Yes No Yes

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.Addendum to HPV2 Chemical Data Adequacy Review (Final ) CASRN 31138

- 65

- 5

A

- 1 Addendum: Test Guideline Source Material to Support Reliability Ratings Assigned to Submitted Studies That Are Considered Inadequate to Satisfy Test Rule Requirements

This addendum provides additional information regarding deficiencies of the acute toxicity to Daphnia study, which is considered inadequate to satisfy the test rule requirement. For each deviation from relevant test guidelines (i.e., standard test guidelines purportedly followed by each study according to the study report), quotes of relevant passages fr om the guidelines are supplied, followed by a brief summary of the quoted material and, where appropriate, an interpretation by a qualified scientist. Emphasis has been added to quotes (in the form of italics) by the reviewer.

Study: Acute Toxicity to D aphnia ( Harris 201 3 ) Relevant Guidelines: The s tudy was conducted according to OECD TG 202 ( equivalent to method C.2 of Commission Regulation (EC) No. 440/2008 ) and OPPTS 850.1010

.
[A] Deviation 1: The pH of the test solution preparations (i.e., solutions containing test substance and dilution water) in the definitive test (and in the range

- finding and post

- study tests) was adjusted to 7 prior to the addition of test organisms. If the test item causes a change in pH that increases the toxicity of the solution, then the toxicity is considered to be the result of the test item. Given that the pH of the test substance is 9.62, the addition of the test substance to the dilution water (consisting of a 50:50 mixture of reconstituted water [pH of 7.8 ± 0.2] and deionized reverse osmosis water [pH not provided]) would be expected to increase the pH. If the resulting pH is outside of the physiologically acceptable range for Daphnia of approximately 6 to 9 (per OECD TG 202), then toxicity may result from t he increase in pH caused by the addition of the test substance. Any toxicity resulting from a change in pH caused by the test substance is considered to be a toxic effect of the test substance. Therefore, by adjusting the pH of test solutions to 7 before exposing the Daphnia to these solutions, the testing laboratory may have altered the toxicity of the test item to Daphnia

. The study results are not representative of the toxicity of the unaltered test substance. OECD TG 20 2 , p. 3, Section “Description of the Method”, Sub

- se ction “Test solutions”, item 13 and Commission Regulation (EC) No. 440/2008, p. 603, Section C.2 “ Daphnia sp. Acute Immobilisation Test”, Sub

- section 1.7 “Description of the Test Method”, item 1.7.4 “Test solutions”, paragraph 2 stat e : “ The test should be carried out without the adjustment of pH. If the pH does not remain in the range 6

- 9, then a second test could be carried out, adjusting the pH of the stock solution to that of the dilution water before addition of the test substance. The pH adjustment should b e made in such a way that the stock solution concentration is not changed to any significant extent and that no chemical reaction or precipitation of the test substance is cau sed. HCl and NaOH are p referred. ” OECD TG 20 2 , p. 4, Section “ Procedure ”, Sub

- se ction “Conditions of Exposure”, “Incubation conditions”, item 19, and Commission Regulation (EC) No. 440/2008, p. 604, Section C.2 “

Daphnia sp. Acute Immobilisation Test”, Sub

- section 1.8 “Procedure”, item 1.8.1 “Conditions of exposure”, sub

- item 1.8.1.3 “Incubation conditions”, paragraph 2 state : “ The test vessels must not be aerated during the test. The test is carried out without adjustment of pH. The daphnids s hould not be fed during the test. ” OPPTS 850.1010, p. 1, Section (b) “Purpose” states: “ This guideline is intended for use in developing data on the acute toxicity of chem ical substances and mixtures (‘chemicals’ )Addendum to HPV2 Chemical Data Adequacy Review (Final ) CASRN 31138

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- 2 subject to environmental effects test regulations. This guideline prescribes an acute toxicity test in which daphnids ( Daphnia magna or D. pulex ) are exposed to a chemical in static and flow

- through systems. The Environmental Protection Agency will use data from this test in assessing the ha zard a chemical may present in the aquatic environment. ”

Summary of Quoted Material : In the initial definitive test, t h e pH of test solutions is not to be adjusted. If the pH of the test solutions in the initial definitive test is not between 6 and 9 after the test substance is added, a second definitive test, in which pH of the stock solution is adjusted to pH of dilution water, could be conducted in addition to the initial definitive test. Data from the acute toxicity to Daph nia test are used by EPA to characterize the hazard of the test substance in aquatic environments. Interpretation: Since natural waters (e.g., streams, lake s , etc.) cannot be reasonably expected to contain chemical substances that would neutralize the pH of the test substance, an acute toxicity to Daphnia test will not provide appropriate information for characterizing hazard of the test substance in natural environments if the pH of the test solution preparations is neutralized prior to the initiation of exposure.

[B] Deviation 2 : Substantially different findings in the range

- finding and definitive tests raise concerns over the reliability of the test methods. In the range

- finding test, 50% immobilization (5/10 daphnids) was observed at 24 hours and 100% immobilization (10/10 daphn ids) was observed at 48 hours at the maximum concentration tested (872 mg a.i./L). In contrast, no effects were observed at 48 hours in the definitive test (maximum test concentration of 889 mg a.i./L) or in the post

- study test (maximum test concentration of 872 mg a.i./L). As presented in the report, similar test procedures were used in the range

- finding, definitive, and post

- study tests (all included adjustment of test solutions to pH 7 prior to the introduction of the daphnids). The investigators cond ucted a post

- study test which confirmed the results of the definitive test, but do not provide a possible explanation for the dissimilar results between the range

- finding and definitive/post

- study tests. OECD TG 20 2 , p. 6, Section “ Data and Reporting ”, Sub

- se ction “Test Report”, item (27) and Commission Regulation (EC) No. 440/2008, p. 606, Section C2 “ Daphnia sp. Acute Immobilization Test”, Sub

- section 3.1 “Test Report” state: “The test report must include the following:”; and, under “Results”, the sixth requirement states : “explanation for any deviation from the Test Guideline [or Testing Method] and whether the deviation affected the test results.” OPPTS 850.1010, p p . 7

- 8 , Section (f) “Reporting” states: “... In addition to the reporting requirements prescribed in 40 CFR Part 792

– Good Laboratory Practice Standards, the reporting of test data should include the following : ” ; and i te m (13) states: “Any deviation from this test guideline and anything unusual about the test, e.g., diluter failure, temperature fluctuations, etc.” ; and i te m (1 4 ) states: “If it is observed that the stability or homogeneity of the test substance cannot be maintained, car e should be taken in the interpretation of the results, and note made that the results may not be reproducible.”

Summary of Quoted Material : The study report should explain any unusual results, such as substantial differences in the results of the range

- finding and definitive tests. Any unusual occurrences during the study that may have affected the results should be reported. If any deviatio ns from the guidelines occurred, the study report must explain these occurrences and discuss whether these deviations impacted the results. If the study results indicate that theAddendum to HPV2 Chemical Data Adequacy Review (Final ) CASRN 31138

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- 3 stability or homogeneity of the test substance cannot be maintained, the stu dy may not be acceptable or the study should be interpreted with caution because the results may not be reproducible. Interpretation: Substantial differences in range

- finding and definitive tests render the results of the definitive study questionable if the study report does not provide (1) a reasonable explanation for the disparity and (2) evidence that the results of the definitive test are more accurate than those of the range

- finding test.

REFERENCE

Harris, S. (2013) Sodium glucoheptonate: Daphn ia sp., 48

- hour acute immobilization test. Performing Laboratory: Harlan Laboratories Ltd., Shardlow, Derbyshire, UK. Study No. 41104695. Sponsor: Harcros Chemicals, Inc., Dalton, GA. Study report available online at http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2007

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- 0719 as of January 14, 2014

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