SRC TR

- 13

- 045

HPV 2 TEST RULE DATA ADEQUACY REVIEW ( REVISED FINAL ) : ACUTE TOXICITY TO FI SH, TOXICITY TO ALGA E, ACUTE MAMMALIAN T OXICITY,

GENE MUTATION, AND C HROMOSOMAL ABERRATIO NS

D

- gluco

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

- , CASRN 31138

- 65

- 5

Prepared by:

Megan Nelson, Amy Nguyen, Jennifer Rayner, Julie Ramage, Lisa Ingerman, 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

- 65

- 5

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. Toxicity to Aquatic Plants

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

D

- 3. Acute Toxicity

................................ ................................ ................................ .................. 18

D

- 4

. Genetic Toxicity

– Gene Mutation

................................ ................................ ................... 21

D

- 5. Genetic Toxicity

– Chromosomal Aberrations

................................ ................................ 25
E. DATA ADEQUACY SUMMARY TABLE

................................ ................................ ........... 30

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

.......... A

- 1Final HPV 2 Chemical Data Adequacy Review CAS RN 31138

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- 5

1 A. SOURCE MATERIAL USED FOR ASSESSMENT

This document evaluates five submission s from Harcros Chemicals, Inc. ( submissions dated from December 18, 2012 to April 24 , 2013) that contain data from toxicity testing of D

- gluco

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

- (CASRN 31138

- 65

- 5) and were received by 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 , 201 4 ).

T he HPV2 test rule requires testing with 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 submissions from Harcros Chemicals, Inc. 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 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 )

 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

- 0531

- 0688 as of Janu ary 14, 2014 ) 2

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

- HQ

- OPPT

2007

- 0531

- 0709 as of January 14, 2014 )

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/opp tintr/exposure/pubs/episuitedl.htm as of January 14, 2014

. 2 Harcros Chemical, Inc. submitted an amendment to this study report on May 22, 2013 via email from A.M. Savini (Harcros Chemicals, Inc.) to J. Schaeffer (EPA) that indicated that formulated concen trations 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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2 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 SIDS toxicity endpoints 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 d ocument and an endpoint 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 summarizi ng 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 Toxicological Data Submitted from December 18 , 201 2 to April 24, 2013 : Data supplied in the submissions identified above are adequate to satisfy the toxicity to plants (algae) ( component of C 1) , acute mammalian toxicity (D), gene mutation (E1)

, and chromosomal aberrations (E2) test requirement s promulgated in the final HPV2 test rule.

However, data submitted for the acute toxicity to fish endpoint (component of C 1) are not considered adequate to satisfy the HPV2 test rule requirement for this endpoint. This study is not adequate for the foll owing reasons:

(1) The pH of test solution preparations in the definitive test was adjusted to 7 prior to the addition of test organisms . This pH adjustment altered the toxicity of the test solutions, as evidenced by the fact that toxicity (3/3 fish killed in extremis after 3 hours of exposure) was observed in the range

- finding test at 889 mg a.i./L without pH adjustment, but no toxicity was observed in the definitive test at 889 mg a.i./L when the pH was adjusted to 7. If the test item causes a change in p H 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 (prepared by dechlorination and sof tening of tap water that had an average pH of 7.497) would be expected to increase the pH. If the resulting pH is outside of the physiologically acceptable range for freshwater fish of approximately 6 to 8 (per OPPTS 850.1075), then toxicity may result fr om 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 be fore exposing the fish to these solutions, the testing laboratory altered the toxicity of the test item to fish. The results produced by the definitive study are not representative of the toxicity of the unaltered test substance. (2) The analytical measurem ents of the test substance ranged from 57 to 184% of nominal concentrations. Test substance concentration measurements for duplicate test solutionFinal HPV 2 Chemical Data Adequacy Review CAS RN 31138

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3 samples varied greatly from one another; differences of up to 2.7

- fold were observed, suggesting that analyt ical techniques for measuring test substance concentrations may not have been reliable and/or accurate . Measured test substance concentrations of “old” test solutions (test medium to which fish had been exposed for 24 hours, sampled just prior to solution renewal at 24, 48, 72, and 96 hours) were up to 105% different from concentrations measured in “fresh” test solutions. No explanation was provided for the variability in measured test substance concentrations. Given the large differences between measure d and nominal concentrations , the endpoint values ( LC 50 , LOEC, and NOEC ) should be based on the mean measured, rath er than nominal, concentrations.

In summary , the submitter needs to repeat the acute toxicity to fish test, implementing the
correct procedures to avoid the deficiencies identified above. The pH of test solutions or stock solutions must not be adjusted after addition of the test substance. EPA recommends that further acute toxicity testing in fish include the conduct of preliminary st udies to determine what additional procedures are necessary for assuring that the analytical method for CASRN 31138

- 65

- 5 can provide accurate and repeatable measurements of the concentrations of this test substance in test solution samples collected during the conduct of a definitive fish acute toxicity

test.

Additional Submitted Toxicological Data : On June 12, 2013, Harcros Chemicals, Inc. submitted an acute toxicity to Daphnia study in response to 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 ) . This study was reviewed in a separate re port (SRC Tech nical Report #13

- 059 ) and was determined to be inadequate to satisfy the acute toxicity to Daphnia test ing requirement promulgated in the final HPV2 test rule. The acute toxicity to Daphnia test needs to be repeated without adjustment of pH in the prepared test solutions.

On August 27, 2013, Harcros Chemicals, Inc. submitted a combined repeated

- dose toxicity study with the reproduction/developmental toxicity screening test in response to th e F1 testing requirement (submitted via email from A.M. Savini [Harcros Chemicals, Inc.] to J. Schaeffer [EPA ] )

. This study was reviewed in a separate report (SRC Technical Report #13

- 071) and was determined to be ad equate 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 Toxicological Endpoints: Data submitted by Harcros Chemicals, Inc. for the acute toxic ity 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 ma mmalian toxicity, repeated

- dose toxicity, gene mutation, chromosomal aberrations, reproductive toxicity, and developmental toxicity endpoints.Final HPV 2 Chemical Data Adequacy Review CAS RN 31138

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4 D . DATA FOR SIDS ENDPOINTS

D

- 1 . Acute Toxicity to Fish

Summary of endpoint: Not a dequate

Study 1: 96

- H our a cute toxicity study in r ainbow t rout ( Harris, 2013 )

Title: Sodium Glucoheptonate: A cute Toxicity to Rainbow Trout ( Oncorhynchus mykiss )

This study is: Adequate Not Adequate

This study is not adequate for the following reasons: (1) The pH of test solution preparations in the definitive test was adjusted to 7 prior to the addition of test organisms . This pH adjustment altered the toxicity of the test solutions, as evidenced by the fact that toxicity (3/3 fish killed in extremis after 3 hours of exposure) was observed in the range

- finding test at 889 mg a.i./L without pH adjustment, but no toxicity was observed in the definitive test at 889 mg a.i./L when the pH was adjusted to 7. If the test item causes a change in pH that incr eases 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 (prepared by dechlorination and softening of t ap water that had an average pH of 7.497) would be expected to increase the pH. If the resulting pH is outside of the physiologically acceptable range for freshwater fish of approximately 6 to 8 (per OPPTS 850.1075), then toxicity may result from the incr ease 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 exposi ng the fish to these solutions, the testing laboratory altered the toxicity of the test item to fish. The results produced by the definitive study are not representative of the toxicity of the unaltered test substance. (2) The analytical measurements of the test substance ranged from 57 to 184% of nominal concentrations. Test substance concentration measurements for duplicate test solution samples varied greatly from one another; differences of up to 2.7

- fold were observed, suggesting that analytical techni ques for measuring test substance concentrations may not have been reliable and/or accurate. Measured test substance concentrations of “old” test solutions (test medium to which fish had been exposed for 24 hours, sampled just prior to solution renewal at 24, 48, 72, and 96 hours) were up to 105% different from concentrations measured in “fresh” test solutions. No explanation was provided for the variability in measured test substance concentrations. Given the large differences between measured and nomin al concentrations, the endpoint values (LC50, LOEC, and NOEC) should be based on the mean measured, rather than nominal, concentrations.

TEST SUBSTANCE

Identity: D

- gluco

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

- (synonym: sodium glucohept onate) ; CASRN 31138

- 65

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

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- 5

5 Remarks: Dark brown liquid; Batch No. 921000100; purity reported as 49 .5% (with 50.5% water) in study report; however, in email s from A.M. Savini (Harcros Chemicals, Inc.) to J. Schaeffer (EPA) on March 5, 2013 and May 14, 2013 , the composition 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 44.02% a.i. 2 (23.4% beta sodium glucoheptonate + 20.62% 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 ( “Fish Acute Toxicity Test, Freshwater and Marine” )

Test type: Acute Fish Toxicity Test (S emi

- S tatic)

GLP compliant ?: Yes

Year study was performed: 201 3

Test species: Rainbow t rout ( Oncorh y nchus mykiss ) obtained from Brow Well Fisheries Limited, located in Hebden , near Skipton, Yorkshire, UK. Fish were acclimatized to test conditions for at least 10 days prior to exposure . The definitive test fish were juveniles that had a mean standard length of 4.3 cm and a mean weight of 0.91 g at the end of the test

.
Analyt ical monitoring: Yes, only at the highest concentration ( 889 mg a

. i

. /L) in the definitive

test

Exposure duration: 96 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

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

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6 Concentrations tested: Nominal c oncentrations: 0, 202, 364, 647, 1131, and 2020 mg test item/ L ; Concentrations adjusted for 44.02 % purity : 0, 89, 160, 284, 498, and 889 mg a.i./L. 1

Test method/conditions remarks: Test water: “Laboratory tap water [average pH 7.497] was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened ( Elga Nimbus 1248D Duplex Water Softener) giving water with a total hardness of approximately 140 mg/L as CaCO 3 .” [Harris 2013, pp. 10]

“At the start of the definitive test the diluent had a particulate matter concentration of 0.1 mg/L, total water hardness of 144 mg/L CaCO 3 , alkalinity of 101.2 mg/L as CaCO 3 , Total Organic Carbon content of less than the limit of quantitation, determined to be 1.0 mg C/L, chemical oxygen demand of 2.09 mg O 2 /L and conductivity of 438 μS/cm. The chlorine content ran ged from 0.000 to 0.091 mg Cl 2 /L.” [Harris 2013, p. 11]

Range

- Finding Test Procedure: Two range

- finding tests were conducted to determine test concentrations for the definitive test. For the range

- finding tests, the test item was dissolved directly in dechlorinated tap water. In the first test, fish were exposed to 2020 mg test item/L (889 mg a.i./L). Mortalities were observed at this concentration and, therefore, a second range

- finding test was conducted

.
In the second range

- finding test, fish were exposed to 2.02, 20.22, or 202.02 mg test item/L (0.89, 8.9, and 89 mg a.i./L, respectively).

“ In the range

- finding test 3 fish were added to each 20 liter test and control vessel and maintained at 13°C to 14° C in a temperature controlled room with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period up to 96 hour s under static test conditions.” [Harris 2013, p. 11]

“The control groups were maintained under identical conditions but not exposed to test item. Each vessel was covered to reduce evaporation. After 3, 6, 24, 48, 72 and 96 hours any mortalities or sub

- lethal effects of exposure were determined by visual inspection of the test fish. ” [Harris 2013, pp. 11

- 12]

“A sample of each test concentration was taken for chemical analysis at 0 and 24 hours in order to determine the stability of the test item under test conditions. All samples were stored at approximately

- 20°C prior to analysi s.” [Harris 2013, p. 12] Measured concentrations were reported only for nominal concentrations of 0.89 to 89 mg a.i./L.

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

- finding samples; full validation was later conducted specifically at the test concentrations employed in the definitive test.” [Harris 2013, p. 28]

1 The study author erroneously report ed equivalent 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%.Final HPV 2 Chemical Data Adequacy Review CAS RN 31138

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7 Definitive Test Procedure: Based on the results of the range

- finding tests

, the concentrations used in the definitive test were 0, 89, 160, 284, 498, and 889 mg a.i./L.

“ For the purpose of the definitive test the test item was dissolved directly in dechlorinated tap

water. Prior to weighing, the test item was boiled to remove any crystals that had formed. ” 1 [Harris 2013, p. 12]

“Amounts of test item (4040, 72721, 12930, 22626 and 40400 mg) were dissolved in dechlorinated tap water and the volume adjusted to 20 liters

... The test media [were] stirred with a flat bladed mixer to ensure complete dissolution. Prior to addition of the test animals, the pH of the test media was adjusted to pH 7.” [Harris 2013, p. 12]

“As in the range

- finding tests, 20 liter glass exposure vessels were used for each test concentration. At the start of the test 10 fish were plac ed in each test vessel at random, in the test preparations. The test vessels were then covered to reduce evaporation and maintained at 13°C to 14°C in a temperature controlled room with a photoperiod of 16 hours light and 8 hours darkness with 20 minute d awn and dusk transition periods for a period of 96 hours. The test vessels received no auxiliary aeration. The fish were not individually identified and received no food during exposure.” [Harris 2013, p. 12]

“The control group was maintained und er identical conditions but not exposed to the test item.” [Harris 2013, p. 12]

“A semi

- static test regime was employed in the test involving a daily renewal of the test preparations to ensure that the concentrations of the test item remained near nominal and to prevent the build up of nitrogenous waste products.” [Harris 2013, p. 12]

“Any mortalities and sub

- lethal effects of exposure were recorded at 3, 6, 24, 48, 72 and 96 hours after the start of exposure. The criteria of death were taken to be the absence of both respiratory movement and response to physical stimulation.” [Harris 2013, p. 13]

“The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the test. The measurements at 0 hours, and after each test media renewal at 24, 48 and 72 hours, represent those of freshly prepared test preparations while the measurements taken prior to each test media renewal, and on termination of the test after 96 hours, represent those of the used or 24

- Hour old test prepar ations

. The pH and oxygen were measured using a Hach HQ30d Flexi handheld meter and the temperature was measured using a Hanna Instruments HI 93510 digital thermometer. The temperature was also monitored in the control hourly using a Testo temperature lo gger. ” [Harris 2013, p. 13]

1 The acute toxicity to fish study report did not provide sufficient information to determine whether the chemical identity of the test substance was changed upon boiling. However, IR scans submitted by Harcros Chemicals, Inc. to EPA on August 12, 2013 (vi a email from A.M. Savini to J. Schaeffer) provide support that the test substance was unaffected by boiling. Although the study report from the acute fish toxicity test does not indicate the length of time for which the test substance was boiled, it appea rs unlikely that the process of boiling the test substance caused a change in its chemical composition.Final HPV 2 Chemical Data Adequacy Review CAS RN 31138

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8 Measurement of Test Concentrations:

“ Water samples were taken from the control and all surviving test groups at 0 (fresh media), 24, 48, [and] 72 (old and fresh media) and 96 (fresh media) hours for quantitative analysis. The samples were stored at approximately

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

“ Duplicate samples were taken and stored at approximately

- 20°C for further analysis if
necessary. Only samples of the NOEC [i.e., 889 mg a.i./L] were analyzed.” [Harris 2013, p. 13]

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

- MS.

RESULTS

Endpoint value(s): Based on nominal concentrations: 96

- hour LC 50 > 2020 mg test item/L ( > 889 mg a.i. /L ) ; 96

- h our LOEC (survival) > 2020 mg test item/L ( > 889 mg a

. i ./L ) ; 96

- h our NOEC (survival) = 2020 mg test item/L ( 889 mg a

. i

. /L )

Results remarks: Range

- Finding Test Results: No mortalities were observed at 0, 0.89, 8.9, or 89 mg a.i./L. At the highest test concentration (889 mg a.i./L): “ After 3 hours all of the fish were observed to have a loss of equilibrium. Due to approach of the substantial severity limit (Animals (Scientific Procedures) Act 1986) these fish were killed and classed as mortalities at the 3 hour time point. ” [Harris 2013, p. 1 7 ]

Analytical measurements of the test substance in the 0.89, 8.9, or 89 mg a.i./L test preparations at 0 and 24 hours ranged from 99% to 126% of nominal concentrations.

Defi nitive T est Results: No mortalities and no sublethal effects were observed in the definitive test at nominal concentrations up to 889 mg a.i./L.

“In the initial range finding test, mortalities were observed at [889 mg a.i./L]... Due to a technical oversight the pH of the test preparations w as not adjusted to within specification. Adjustments to pH were made during the definitive test and hence it was considered likely that the mortalities observed in the initial range finding test were due to a pH effect rather than a true toxic effect.” 1 [Harris 2013, p. 15]

Throughout the test, the 89 and 160 mg a

. i

. /L test preparations were observed to be light yellow solutions

, increasing in color with concentration, and the 284, 498, and 889 mg a.i. /L test preparations were observed to be dark brown solutions

, increasing in color with concentration

.
Physico

- Chemical Measurements: “The water temperature which was

... recorded in the control vessel approximat ely every hour

1 While the study author considers the pH effect to not be a “true” toxic effect, EPA disagrees. Any toxicity resulting from a change in pH caused by t he test substance is considered by EPA to be a true toxic effect.Final HPV 2 Chemical Data Adequacy Review CAS RN 31138

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9 using a Testo temperature logger

... ranged from 12 ° C to 14 ° C.” 1 [Harris 2013, p. 15]

Daily water temperature measurements , measured every 24 hours in old and fresh test media at all test concentrations, ranged from 13 to 14 ° C . The pH was 6.9

- 7.4 , while dissolved oxygen

was 8.3

- 11.4 mg/L ( 81

- 111 % saturation).

“The oxygen concentration in some of the test vessels was observed to have an air saturation value (ASV) in excess of 100%. This was considered to be due to the presence of microscopic air bubbles in the media super

- saturating the diluent and was considered to not have had an impa ct on the outcome o r integrity of the test as no adverse effects were observed in the control group.” [Harris 2013, p. 15]

Verification of Test Concentrations: “Analysis of the ... [ second set of duplicate samples taken for analysis of test substance

conce ntration] at 0, 24, 48, 72 and 96 hours showed measured test concentrations [at the highest test concentration] to range from 91% to 114% of nominal [with one exception] and so the results are based on nominal test concentrations only. The fresh test prep aration at 48 hours was measured to be 184% of nominal, however, as the corresponding old preparation at 72 hours was near nominal it is considered that this result is anomalous and has no impact on the outcome of the test.” [Harris 2013, pp. 15

- 16]

The first set of duplicate samples analyzed for test substance concentration showed measured test concentrations ranging from 57 to 95% of nominal concentrations. The study author did not provide an explanation for the discrepancy between the two sets of samp les.

CONCLUSIONS

The 96

- hour LC 50 for rainbow trout was greater than 889 mg a

. i

. /L. Additionally, the 96

- h our LOEC and NOEC were greater than 889 mg a

. i

. /L and equal to 889 mg a

. i

. /L, respectively.

STUDY RELIABILITY

[ 3 ] Not reliable. This study is not reliable for the following reasons: (1) The pH of test solution preparations in the definitive test was adjusted to 7 prior to the addition of test organisms. This pH adjustment altered the toxicity of the test solutions, as evidenced by the fact that toxicity (3/3 fish killed in extremis after 3 hours of exposure) was observed in the range

- finding test at 889 mg a.i./L without pH adjustment, but no toxicity was observed in the definitive test at 889 mg a.i./L when the pH was adjusted to 7. 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 (prepared by dechlorination and softening of tap wat er that had an average pH of 7.497) would be expected to increase the pH. If the resulting pH is outside of the physiologically acceptable range for freshwater fish of approximately 6 to 8 (per OPPTS

1 Contradicting information is shown in Figure 1 of Harris (2013) , which shows continuous temperature measurements ranging from approximately 10 to 14 °C.Final HPV 2 Chemical Data Adequacy Review CAS RN 31138

- 65

- 5

10 850.1075), then toxicity may result from the increase i n 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 fish to these solutions, the testing laboratory altered the toxicity of the test item to fish. The results produced by the definitive study are not representative of the toxicity of the unaltered test substance. (2) The analytical measurements of the test substance ranged from 57 to 184% of nominal concentrations. Test substance concentration measurements for duplicate test solution samples varied greatly from one another; differences of up to 2.7

- fold were observed

, suggesting that analytical techniques for measuring test substance concentrations may not have been reliable and/or accurate . Measured test substance concentrations of “old” test solutions (test medium to which fish had been exposed for 24 hours, sampled just prior to solution renewal at 24, 48, 72, and 96 hours) were up to 105% different from concentrations measured in “fresh” test solutions. No explanation was provided for the variability in measured test substance concentrations. Given the large differences between measured and nominal co ncentrations , the endpoint values ( LC 50 , LOEC, and NOEC ) should be based on the mean measured, rather than nominal, concentrations.

REFERENCE

Harris, S. ( 2013 ) Sodium g lucoheptonate: Acute toxicity to rainbow trout ( Oncorhynchus mykiss )

. Performing Laboratory: Harlan Laboratories Ltd.

, Shardlow, Derbyshire, UK. Study No. 41104694. 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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11 D

- 2 . Toxicity to Aquatic Plants

Summary of endpoint: Adequate

Study 1: Toxicity to Pseudokirchneriella subcapitata ( Vryenhoef, 2013)

Title: Sodium Glucoheptonate: Algal Inhibition Test

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: Dark brown liquid; Batch No. 921000100; purity reported as 49 .5% (with 50.5% water) in study report; however, in email s from A.M. Savini (Harcros Chemicals, Inc.) to J. Schaeffer (EPA) on March 5, 2013 and May 14, 2013 , the composition of the test substance was reported as f

ollows: 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 44.02% a.i. 2 (23.4% beta sodium glucoheptonate + 20.62% alpha sodium glucoheptonate ).

METHOD

Method/guideline followed: OECD TG 201 (2006 ; “Freshwater Alga and Cyanobacteria, Growth Inhibition Test” ) referenced as Method C.3 of Commission Regulation (EC) No. 761/2009 and OPPTS 850.5400 ( “Algal Toxicity, Tiers I and II” )

Test type: Toxicity Test to Freshwater Algae (Static Conditions)

1 30090

- 50

- 3 is not a valid CAS nu mber 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

- 65

- 5

12 GLP compliant?: Yes

Year study was performed: 201 2

Test species: Green algae ( Pseudokirchneriella subcapitata )

, strain CCAP 278/4

, were obtained from Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd. in Argyll, Scotland.

Analytical monitoring: Yes (at 0 and 96 hours)

Exposure duration: 96 hours

Concentrations tested: Nominal concentrations adjusted for 44. 02% purity: ~ 0, 8.9, 28, 89, 284

, and 889 mg a.i./L 1

Test method/conditions remarks:

The culture medium used in both the range

- finding and definitive tests had a hardness of 18 mg/L as CaCO 3 and a Total Organic Carbon (TOC) content of less than 1.0 mg C/L.

Range

- finding T est Procedure: In t he range

- finding test

, Pseudokirchneriella subcapitata cells were exposed to a series of nominal test concentrations of 0.89, 8.9, 89, and 889 mg a.i./L for 96 hours. 2

“ The test was conducted in 250 mL glass conical flasks each containing 100 mL of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were used for each control and test concentration. Th e test item was dissolved directly in culture medium. ” [ Vryenhoef 2013, p. 11]

To prepare the stock solution of 889 mg a.i./L, 1010 mg of test item was dissolved in culture medium and the volume adjusted to 500 mL

. A series of dilutions was made to give solutions of 0.89, 8.9, and 89 mg a.i./L

. An aliquot (450 mL) of each solution was inoculated with algal suspension (9.7 mL)

.

“ The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity. ” [ Vryenhoef 2013, p. 11]

“ The control group was maintained under identical conditions but not exposed to the test item. ” [ Vryenhoef 2013, p. 11]

1 The study author reported nominal concentrations of 0, 10, 32, 100, 320, and 1000 mg a.i./L

, which were calculated based on a purity of 49.5% (water content of 50.5%) . The correct test substance purity was 44.02%. The concentrations presented in the text above have been corrected for 44.02% purity.

2 The study author reported nominal concentrations of 1.0, 10, 100 and 1000 mg a.i./L , which were calculated based on an incorrect purity of 49.5% (water content of 50.5%). 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

- 65

- 5

13 “ At the start of the range

- finding test a sample of each test and control culture was removed and the cell density determined using a Coulter ® Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron ® Version 2 incubator) at 24 ± 1 ° C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380

– 730 nm) and constantly shaken at approximately 150 rpm for 96 hours. ” [ Vryenhoef 2013, p. 11]

“ After 96 hours the cell density of each flask was determined using a Coulter ® Multisizer Particle Counter.” [ Vryenhoef 2013, p. 12]

“ A sample of each test concentration was taken for chemical analysis at 0 and 96 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.” [ Vryenhoef 2013, p. 12]

“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 to be employed in the definitive test.” [ Vryenhoef 2013, p. 43 ]

Definitive T est Procedure: Based on the results of the range

- finding test, concentrations of 8.9, 28, 89, 284, and 889 mg a.i./L were chosen for the definitive test. 1

To prepare the 889 and 284 mg a.i ./L stock solutions, respectively, 2020 and 646 mg of the test item were dissolved in 1 L of culture medium. A series of dilutions was made from these stock solutions to give solutions of 8.9, 28, and 89 mg a.i./L

. An aliquot (900 mL) of each solution

wa s inoculated with algal suspension (13.5 mL)

.

“ The stock solutions and each of the prepared concentrations were inverted several times to

ensure adequate mixing and homogeneity.” [ Vryenhoef 2013, p. 12]

“ The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 96 hours. ” [ Vryenhoef 2013, p. 12]

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

- MS.

“ As in the range

- finding test 250 mL glass conical flasks were used. Six flasks each containing 100 mL of test preparation were used for the control and three flasks each containing 100 mL

were used for each treatment group.” [ Vryenhoef 2013, p. 12]

” The control group was maintained under identical conditions but not exposed to the test item. ” [ Vryenhoef 2013, p. 12]

1 The study author reported nominal concentrations of 10, 32, 100, 320 and 1000 mg a .i./ L , which were calculated based on an incorrect purity of 49.5% (water content of 50.5%). The correct test substance purity was 44.02%. The concentrations presented in the text above have been co rrected for 44.02% purity.Final HPV 2 Chemical Data Adequacy Review CAS RN 31138

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- 5

14 “ Pre

- culture conditions gave an algal suspension in log phase growth characterized by a cell density of 6.68 x 10 5 cells per mL. Inocula tion of 900 mL of test medium with 13.5 mL of this algal suspension gave an initial nominal cell density of 1 x 10 4 cells per mL and had no significant dilution effect o n the final test concentration.” [ Vryenhoef 2013, p. 12]

“ The flasks were plugged wit h polyurethane foam bungs and incubated (INFORS Multitron ® Version 2 incubator) at 24 ± 1 ° C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380

– 730 nm) and constantly shaken at approximately 150 rpm for 96 hours. ” [ Vryenhoef 2013, pp. 12

- 13]

“ Samples were taken at 0, 24, 48, 72 and 96 hours and the cell densities determined using a Coulter ® Mu ltisizer Particle Counter.” [ Vryenhoef 2013, p. 1 3 ]

“At the request of the Sponsor, at the start of the test the pH of the control and test preparations was adjusted to pH 7. The pH of the control and each test preparation was determined at initiation of the test and after both 48 and 96 hours exposure... The temperature within the incubator was recorded hourly... ” [ Vryenhoef 2013, p. 13]

Re

- growth T est P rocedure: “A re

- growth test was performed after 96 hours to determine the algicidal or algistatic effect of the test item. Aliquots were removed from each replicate control and test culture (0.25 mL and 0.50 mL respectively) and the replicates pooled. Fresh sterile culture medium (100 mL) was added to each to ensure that the test concentration was reduced to below the inhibit ing level. ” [ Vryenhoef 2013, p. 13]

“ The flasks were plugged with polyurethane foam bungs are incubated (INFORS Multitron ® Version 2 incubator) at 24 ± 1 ° C under continuous illumination (intensity approximately 7000 lux) and constantly shaken at approximately 150 rpm for 120 hours.” [ Vryenhoef 2013, p. 13]

Statistical Analysis: “One way analysis of variance incorporating Bartlett’s test for homogeneity of variance

... and Dunnett’s multiple comparison procedure for comparing several treatments with a control

... was carried out on the growth rate, yield and biomass integral data after 72 and 96 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999

- 2001).” [ Vryenhoef 2013, pp. 15

- 16]

Validation Criteria: The results of the test were considered valid if the fol lowing criteria were met:

 The cell concentration in control cultures increased by a factor of at least 16 after 72 hours and a factor of at least 100 after 96 hours.

 The mean coefficient of variation of the section

- by

- section specific growth rates in

contr ols during 0

- 72 hours did not exceed 35%.

 The coefficient of variation of the average specific growth rate in controls did not exceed 7% after 72 hours.Final HPV 2 Chemical Data Adequacy Review CAS RN 31138

- 65

- 5

15  The coefficient of variation of the cell density measurements in controls did not exceed 20% after 72 and 96 hours.

RESULTS

Endpoint value(s): The following results were determined from the data based on nominal concentrations: 1

Growth Rate: 72

- hour E r C 50 = 454 mg a

. i

. /L * 72

- hour NOEC = 28 mg a

. i

. /L; 72

- hour LOEC = 89 mg a

. i

. /L 96

- hour E r C 50 = 703 mg a

. i

. /L * 96

- hour NOEC = 89 mg a

. i

. /L; 96

- hour LOEC = 284 mg a

. i

. /L

Yield: 72

- hour E y C 50 = 133 mg a

. i

. /L (95% confidence limits: 107

- 169 mg a

. i

. /L) 72

- hour NOEC = 28 mg a

. i

. /L; 72

- hour LOEC = 89 mg a

. i

. /L 96

- hour E y C 50 = 196 mg a

. i

. /L (95% confidence limits: 1 6 0

- 2 31 mg a

. i

. /L) 96

- hour N OEC = 8.9 mg a

. i

. /L; 96

- hour LOEC = 28 mg a

. i

. /L

Biomass: 72

- hour E b C 50 = 142 mg a

. i

. /L (95% confidence limits: 1 25

- 1 69 mg a.i./L) 72

- hour NOEC = 28 mg a

. i

. /L; 72

- hour LOEC = 89 mg a

. i

. /L 96

- hour E b C 50 = 1 6 0 mg a

. i

. /L (95% confidence limits: 1 25

- 187 mg a.i./L) 96

- hour N OEC = 28 mg a

. i

. /L; 96

- hour LOEC = 89 mg a

. i

. /L

* 95% Confidence limits not calculable

Results remarks:

Range

- F inding T est Results: No effects on growth rate were observed at 0.89 and 8.9 mg a.i./L. Growth rates were reduced at 89 and 889 mg a.i./L (4 and 49% inhibition, respectively) compared to controls. Mean i nhibition of yield and biomass was 3, 12, 22, and 93% at test concentrations of 0.89, 8.9, 89, and 889 mg a.i./L, respectively , relative to controls

.

Measured test substance concentrations of the 8.9, 89, and 889 mg a.i./L test solutions at 0 and 96 hours range d from 87% to 129% of nominal concentrations. Results were not reported for the 0.89 mg a.i./L test solutions.

Definitive T est Results: After 72 hours, mean inhibition of growth rate, relative to controls, was 1, 1, 7, 32, and 75% at test concentrations of 8.9, 28, 89, 284, and 889 mg a.i./L, respectively; effects on growth rate

1 The study author reported endpoint values based on an incorrect purity of 49.5% (water content of 50.5%). The correct test substance purity was 44.02%. The endpoint values presented in the text above have been corrected for 44.02% purity.Final HPV 2 Chemical Data Adequacy Review CAS RN 31138

- 65

- 5

16 were signif

icant (P < 0.5) at concentrations ≥ 89 mg a.i./L after 72 hours . After 96 hours, mean inhibition of growth rate, relative to controls, was

- 1, 3, 4, 18, and 60% at test concentrations of 8.9, 28, 89, 284, and 889 mg a.i./L, respectively; effects on growth rate were significant (P < 0.5) at concentrations ≥ 284 mg a.i./L after 96 hours

.
After 72 hours, mean inhibition of yield, relative to controls, was 6, 7, 29, 82, and 98% at test concentrations of 8.9, 28, 89, 284, and 889 mg a.i./ L, respectively; effects on yield were significant (P < 0.5) at concentrations ≥ 89 mg a.i./L after 72 hours. After 96 hours, mean inhibition of yield, relative to controls, was

- 8, 13, 22, 63, and 97% at test concentrations of 8.9, 28, 89, 284, and 889 m g a.i./L, respectively; effects on yield were significant (P < 0.5) at concentrations ≥ 28 mg a.i./L after 96 hours.

After 72 hours, mean inhibition of biomass, relative to controls, was 7, 9, 26, 81, and 97% at test concentrations of 8.9, 28, 89, 284, an d 889 mg a.i./L, respectively; effects on biomass were significant (P < 0.5) at concentrations ≥ 89 mg a.i./L after 72 hours. After 96 hours, mean inhibition of biomass, relative to controls, was 1, 10, 26, 74, and 97 % at test concentrations of 8.9, 28, 8 9, 284, and 889 mg a.i./L, respectively; effects on biomass were significant (P < 0.5) at concentrations ≥ 89 mg a.i./L after 96 hours.

The test was considered valid because: the cell concentration in controls increased by a factor of 160 after 72 hours and by a fa ctor of 239 after 96 hours, the mean coefficient of variation for section

- by

- section specific growth rate for controls over 0

- 72 hours was 9%, the coefficient of variation for average specific growth rate for controls after 72 hours was 1%, and the coefficient of variation of the cell density values in replicate control cultures was 12% after 72 hours and 3% after 96 hours.

Microscopic examination of control and test cultures at 8.9, 28, 89, and 284 mg a.i./L after 96 hours revealed no abnormalities ; however

, cell debris was observed to be present at 889 mg

a

. i

. /L.”

T est cultures were observed to range from clear

, colorless solutions at 8.9 mg a

. i

. /L to amber

colored solutions at 889 mg a

. i

. /L. After 96 h our s, all test cultures except the 889 mg a.i./L cultures were observed to be green dispersions

, whil e the 889 mg a

. i

. /L test cultures were amber/green dispersions

.
During the test, temperature was 24 ± 1 °C and pH ranged from 7.0 to 8.2. Prior to pH adjustment to 7.0 before t he test began, the pH of test solutions was 7.7, 7.2, 7.4, 8.7, 9.3, and 9.6 at concentrations of 0, 8.9, 28, 89, 284, and 889 mg a.i./L, respectively

.
“Analysis of the test preparations at 0 and 96 hours showed measured test concentrations to range from 93% to 105% of nominal and so the results are based on nominal test concentrations only.” [ Vryenhoef 2013, p. 7]

Regrowth T est Results: Regrowth was observed at 8.9, 28, 89, and 284 mg a

. i

. /L after 48 hours and at 889 mg a

. i

. /L after 120 hours

, indicat ing that the test item was algistatic

.Final HPV 2 Chemical Data Adequacy Review CAS RN 31138

- 65

- 5

17 CONCLUSIONS

The following results were determined from the data based on nominal concentrations:

The 72

- hr EC 50 was found to be 454 mg a

. i

. /L for growth rate

, 133 mg a

. i

. /L for yield , and 142 mg a.i./L for biomass

. The 72

- hour NOEC and LOEC for growth rate were 28 and 89 mg a

. i

. /L, respectively. The 72

- hour NOEC and LOEC for yield were 28 and 89 mg a

. i

. /L, respectively. The 72

- hour NOEC and LOEC for biomass were 28 and 89 mg a

. i

. /L, respectively

.

The 96

- hr EC 50 was found to be 703 mg a

. i

. /L for growth rate

, 196 mg a

. i

. /L for yield , and 160 mg a.i./L for biomass

. The 96

- hour NOEC and LOEC for growth rate were 89 and 284 mg a

. i

. /L, respectively. The 96

- hour NOEC and LOEC for yield were 8.9 and 28 mg a

. i

. /L, respectively. The 96

- hour NOEC and LOEC for biomass were 28 and 89 mg a

. i

. /L, respectively

.

STUDY RELIABILITY

[ 2 ] Reliable with restrictions. The adjustment of the pH of test solution preparations to 7 at the start of the test may have altered the toxicity of the test solutions . However, the results of this test appear to be acceptable given the following: (1) Algae are relatively tolerant of a basic pH, such as that resulting from addition of the test substance to culture media; the effect of pH ( at the levels resulting from test substance addition) on algal growth is not expected to appreciabl y affect the toxicity of the test solutions to algae. (2) The toxicity observed in the range

- finding test, in which pH of test solutions appeared to be unad justed, was similar to the toxicity observed in the definitive test.

REFERENCE

Vryenhoef, H

. ( 2013 ) Sodium gl ucoheptonate: Algal inhibition t est

. Performing Laboratory: Harlan Laboratories Ltd.

, Shardlow, Derbyshire, UK. Study No. 41104696. Sponsor: Harcros Chemica ls, Inc.

, Dalton, GA.

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

- 65

- 5

18 D

- 3 . Acute Toxicity

Summary of endpoint: Adequate

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

Title: Sodium Glucoheptonate: A cute Oral Toxicity in the Rat

– Up

- A nd

- Down Procedure

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: Dark brown liquid; Batch No. 921000100; purity reported as 49 .5% in study report; however, in email s from A.M. Savini (Harcros Chemicals, Inc.) to J. Schaeffer (EPA) on March 5, 2013 and May 14 , 2013 , the composition 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 44.02% a.i. 2 (23.4% beta sodium gl ucoheptonate + 20.62% alpha sodium glucoheptonate ).

METHOD

Method/guideline followed: OECD TG 425 (2008), "Acute Oral Toxicity

- Up

- and

- Down

- Procedure (UDP) ”

Test type: Acute Ora l

– Ra t

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.ni h.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

- 65

- 5

19 GLP com pl iant?: Yes, with exception: “No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. The test item was formulated within two hours of being applied to the test system; it is assumed that the formulation was stable for this duration. This exception is considered not to affect the purpose or integrity of the study.” [ Sanders 2013, p. 3]

Year study performed: 2012

Test species: Female Wistar (RccHan TM :WIST strain) rats were supplied by Harlan Laboratories UK Ltd., located in Oxon, UK. At the start of the study, the test animals were 8 to 12 weeks old and weighed 151 to 167 grams.

Number of animals/sex/dose: 5 females/dose

Route of administration: Oral (Gavage)

Vehicle: Distilled water

Doses/concentrations tested: Nominal doses : 354, 1112

, and 4040 mg test item /kg

- bw ; Doses adjusted for % active ingre dient (based on 44.02% purity): 15 6 , 49 0, and 1778 mg a.i./kg

- bw. 1

S tatistical methods used: Not applicable

Test method/conditions remarks: “All animals were dosed once only by gavage, using a metal cannula attached to a graduated syringe. The volume administered to each animal was calculated according to the fasted bodyweight at the time of dosing. Treatment of animals was sequential. Suff icient time (at least 48 hours) was allowed between each individual animal to confirm the outcome of the previously dosed animals.” [ Sanders 2013, pp. 8

- 9]

The AOT425 Statistical Program was used to determine dose progression. The first test animal

was administered a single dosage of 354 mg test item/kg

- bw (corresponding to 156 mg a.i./kg

bw). Following the first test animal, test animals 2, 3, 4, and 5 received sequential doses of 1112, 4040, 4040 and 4040 mg test item/kg

- bw, respectively (490, 1778, 1 778, and 1778 mg a.i./kg

- bw, respectively). Testing was complete when test animals 3, 4, and 5 survived the maximum dose level of 4040 mg test item/kg

- bw (1778 mg a.i./kg

- bw).

“The animals were observed for deaths or overt signs of toxicity 1⁄2, 1, 2 and 4 hours after dosing and subsequently once daily for fourteen days.” [ Sanders 2013, p. 9]

“Individual body weights were recorded prior to dosing and seven and fourteen days aft er treatment.” [ Sanders 2013, p. 9]

1 The study author erroneously reported equival ent concentrations of 175, 550, and 2000 mg a.i./kg

- bw, respectively, based on a n incorrect purity of 49.5%. The correct test substance purity was 44.02%.Final HPV 2 Chemical Data Adequacy Review CAS RN 31138

- 65

- 5

20 “At the end of the observation period the animals were killed by cervical dislocation. All animals were subjected to gross pathological examination. This consisted of an external examination and opening of the abdomi nal and thoracic cavities for examination of major organs. The appearance of any macroscopic abnormalities was recorded. No tissues were retained.” [ Sanders 2013, p. 9]

RESULTS

Endpoint value(s ): LD 50 > 4040 mg test item/kg

- bw (> 1778 mg a.i./kg

- bw)

Results remarks: At termination of this study, no mortalities had been observed.

Clinical Observations: “Hunched posture was noted in the animal treated at a dose level of 1112 mg [test item]/kg [490 mg a.i./L]... No other signs of systemic toxicity w ere noted.” [ Sanders 2013, p. 10]

Bodyweight: “Animals showed expected gains in bodyweight over the study period, except for one animal treated at a dose level of 4040 mg [test item]/kg [1778 mg a.i./L]... which showed expected gain in bodyweight during t he first week but no gain in bodyweight during the second week.” [ Sanders 2013, p. 10]

Necropsy: “No abnormalities were noted at necropsy.” [Sanders 2013, p. 10]

CONCLUSIONS

“The acute oral median lethal dose (LD 50 ) of the test item in the female Wistar strain rat was found to be greater than 4040 mg [test item] /kg bodyweight [1778 mg a.i./L]... ” [ Sanders 2013, p. 11]

STUDY RELIABILITY

[2 ] Reliable with restrictions. The maximum dose of 1778 mg a.i./kg

- bw employed in this study is somewhat less than the maximum dose of 2000 mg/kg

- bw recommended by the test guideline specified by the HPV2 test rule (40 CFR 799.9110(d)(1)(i)(A) or OECD TG 425) in the absence of mortality.

REFERENCES

Sanders, A. (2013) Sodium glucoheptonate: Acute oral toxicity in the rat

– up

- and

- down procedure

. Performing Laboratory: Harlan Laboratories Ltd.

, Shardlow, Derbyshire, UK. Project No. 41104687. Sponsor: Harcros Chemicals, Inc.

, Dalton, GA.

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

- 65

- 5

21 D

- 4 . Genetic Toxicity

– Gene Mutation

Summary of endpoint: Adequate

Study 1: Reverse mutation assay in Salmonella typhimurium and Escherichia coli (Thompson, 2012 )

Title: Sodium glucoheptonate: Reverse mutation assay “Ames test” using Salmonella typhimurium and Escherichia coli

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 : Dark brown liquid; Batch No. 921000100; purity reported as 50.5% (with 49.5% water) in study report; however, in an email from A.M. Savini (Harcros Chemicals, Inc.) to J. Schaeffer (EPA) on March 5, 2013, the composition of the test substance was report ed 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 ht tp://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 44.02% a.i. 2 (23.4% beta sodium glucoheptonate + 20.62% alpha sodium glucoheptonate).

1 30090

- 50

- 3 is not a valid CAS number according to the U.S. National Library of Medicine’s ChemIDpl us 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

- 65

- 5

22 METHOD

Method/guideline followed: OECD TG 471 (“ Bacterial Reverse Mutation Test”) ; EU Method B13/14 of Commission Regulation (EC) number 440/2008; and the USA EPA (TSCA) OPPTS harmonized guidelines.

Test type: Bacterial Reverse Mutation Test

GLP compliant?: Yes, with exception: “No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. The test item was formulated within four hours of it being applied to the test system. It is assumed that the formu lation was stable for this duration. This exception is considered not to affect the purpose or integrity of the study.” [Thompson, 2012, p. 3]

Year study performed: 2012

Test species: Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100; Escherichia coli strain WP2 uvrA

Metabolic activation: With and without ( rat liver S9 homogenate )

Concentrations tested: 44, 131, 436, 1308, and 4358 μg a.i./plate 1

Statistical methods used: According to Mahon, G.A.T. et al. (1989) Analysis of data from microbial colony assays. In: Statistical Evaluation of Mutagenicity Test Data, UKEMS sub

committee on guidelines for mutagenicity testing, (Kirkland, D. J., Ed.), Cambridge University Press Report , 26

- 65.

Test method/conditions remarks: The mutagenicity assay was conducted by exposing Salmonella typhimurium tester strains TA1535, TA1537, TA98, and TA100, and Escherichia coli strain WP2 uvrA with and without an exogenous metabolic activating system, rat liver homogenate. S9 fractions were derived from rats induced orally with phenobarbitone/β

naphthoflavone orally for three days. Sterile distilled water was used as a vehicle for the test

su bstance. All test solutions were used within 4 hours of preparation. The concentrations for the first experiment were determined by a preliminary toxicity assay, in which nominal tested concentrations were reported as 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 5 00, 1500, and 5000 μg/plate 2 in S. typhimurium strain TA100 and E. coli strain WP2 uvrA using the plate incorporation method with and without metabolic activation. No toxicity was observed in the preliminary toxicity test.

1 The study author reported concentrations of 0, 50, 150, 500, 1500, and 5000 μg/plate , which were calculated by adjusting for a purity of 50.5% (water content of 49.5%). The correct test substance purity was 4 4.02%. The concentrations presented in the text above have been corrected for 44.02% purity.

2 The study report does not indicate whether the concentrations reported for the preliminary toxicity test are adjusted for % purity of the test substance.Final HPV 2 Chemical Data Adequacy Review CAS RN 31138

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23 I n experiment 1, concentrations of 44, 131, 436, 1308, and 4358 μg a.i./plate 1 were assayed in triplicate for each tester strain, using the direct plate incorporation method. Experiment 2 tested the same concentrations as experiment 1, in triplicate for each tester strain, using the pre

incubation test method.

All of the plates we re incubated at 37°C for ~48 hours and revertant colonies were counted using an automated colony counter.

A solvent treatment group was used as the vehicle control; positive controls without S9

- mix included N

- ethyl

- N’

- nitro

- N

- nitrosoguanidine (2 μg/plate for WP2 uvrA, 3 μg/plate for TA100, and 5 μg/plate for TA1535), 9

- aminoacridine (80 μg/plate for TA1537), and 4

- nitroquinoline

- 1

- oxide (0.2 μg/plate for TA98). Additionally, 2

- aminoanthracene (1 μg/plate for TA100, 2 μg/plate for TA1535 and TA1537, and 10 μg/plate for WP2 uvrA ) and benzo(a)pyrene (5 μg/plate for TA98), which are non

- mutagenic in the absence of metabolizing enzymes, were used as positive controls with S9

- mix.

The test article was evaluated as positive if: a dose

- related increase in mutant frequency occurred over the dose range tested, a reproducible increase occurred at one or more concentrations, biological relevance against in

- house historical ranges was seen, statistical analysis of data as determined by UKEMS proved significant, and/or an increase of greater than

two

- fold occurred as compared to the concurrent solvent control for any tester strain. The test item response was evaluated as negative if the above criteria were not met. An equivocal result would be reported when an experim ent did not give a clear positive or negative result.

RESULTS

Genotoxicity result: Negative in the presence and absence of metabolic activation

Cytotoxic concentration (if applicable): > 4358 μg a.i./plate

Results remarks: In the preliminary toxicity test, the test item was non

- toxic to both strains of bacteria used (TA100 and WP2 uvrA ).

No visible reduction in the growth of the bacterial background lawn was observed at any concentration tested in any of the tests. No test item precipitate was observed at any of the concentrations tested in either the presence or absence of S9

- mix.

“No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or [by either] exposure method.” [Thompson, 2012, p. 16]

1 The study author reported concentrations of 0, 50, 150, 500, 1500, and 5000 μg/plate , which were calculated by adjusting for a purity of 50.5% (water content of 49.5%). The correct test substance purity was 44.02%. The concentrations presented in the text ab ove have been corrected for 44.02% purity.Final HPV 2 Chemical Data Adequacy Review CAS RN 31138

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24 Solvent controls tested concurrently responded appropriately. Positive controls induced marked increases in the frequency of revertant colonies, c onfirming the activity of the S9

- mix and the sensitivity of the bacterial strains.

CONCLUSIONS

“The test item, Sodium Glucoheptonate, was considered to be non

- mutagenic under the conditions of this test.” [Thompson, 2012, p. 16]

STUDY RELIABILITY

[2] Reliable with restrictions. The maximum concentration of 4358 μg a.i./plate employed in this study is somewhat less than the maximum concentration of 5000 μg/plate recommended for soluble non

- cytotoxic substances by t est guidelines 40 CFR 799.9510 (guideline specified by the HPV2 test rule) and OECD TG 471 (guideline followed according to the study report).

REFERENCES

Thompson, PW. (2012) Sodium glucoheptonate: Reverse mutation assay “Ames test” using Salmonella typhimurium and Esc herichia coli . Performing Laboratory: Harlan Laboratories Ltd., Shardlow, Derbyshire, UK. Project number: 41104688. Sponsor: Harcros Chemicals Inc., Dalton, GA. 1

Text in quotes is taken directly from Thompson (2012).

1 Harcros Chemical, Inc. submitted an amendment to this study report on May 22, 2013 via email from A.M. Savini (Harcros Chemicals, Inc.) to J. Schaeffer (EPA) 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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25 D

- 5 . Genetic Toxicity

– Chromosomal Aberrations

Summary of endpoint: Adequate

Study 1: In vivo mouse micronucleus assay ( Flanders, 2013)

Title : Sodium Glucoheptonate: Micronucleus Test in the Mouse

The study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: D

- gluco

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

- (synonym: sodium glucoheptonate); CASRN 31138

- 65

- 5

Remarks: Dark brown liquid; Batch No. 921000100; purity reported as 50.5% (with 49.5% water) in study report; however, in email s from A.M. Savini (Harcros Chemicals, Inc.) to J. Schaeffer (EPA) on March 5, 2013 and May 14, 2013 , the composition of the test substance was reported as f

ollows: 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 44.02% a.i. 2 (23.4% beta sodium glucoheptonate + 20.62% alpha sodium glucoheptonate ).

METHOD

Method/ guideline followed: OECD TG 474 (1997 ; “Mammalian Erythrocyte Micronucleus Test”) , Method B12 of Commission Regulation (EC) No. 440/2008 (2008), OPPTS 870.5395 (1998 ; “ In vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay ” )

, and Japa nese METI/MHLW/MAFF Guidelines

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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26 Test type: In vivo Mouse Micronucleus Assay

GLP com pl iant?: Yes, with exception: “No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. The test item was formulated within two hours of it being applied to the test system; it is assumed that the formulat ion was stable for this duration. This exception is considered not to affect the purpose or integrity of the study.” [ Flanders 2013, p. 3]

Year study performed: 2012

Test species: Albino Hsd: ICR (CD

- 1 ® ) mice were obtained from Harlan Laboratories UK Ltd., located in Oxon, UK. Prior to testing, the test animals were approximately 6 to 10 weeks old and weighed 21 to 30 grams.

Metabolic activation: Not applicable

Number of animals/sex/dose: 7 males/dose

Route of administration: Range

- finding: o ral (gavage) or intraperitoneal (i.p.) ; Definitive: intraperitoneal

Vehicle: Phosphate buffered saline (PBS)

Doses/c oncentrations tested: 0 (vehicle only), 436

, 872

, and 1743 mg a.i. /kg

- bw 1

Statistical methods used: “All data were statistically analysed using appropriate statistical methods as recommended by the UKEMS Sub

- committee on Guidelines for Mutagenicity Testing Report, Part III (1989). The data was analysed following a transformation using Student’s t

- test (two tailed) and any significant results were confirmed using the one way analysis of variance.” [Flanders 2013, p. 1 3 ]

Test method/conditions remarks:

“ For the purpose of this study the test item was freshly prepa red as required as a solution at the appropriate concentration in phosphate buffered saline (PBS). ” [ Flanders 2013, p. 8]

Range

- Finding Study Procedure: “A range

- finding toxicity test was performed to determine a suitable dose level and route of administration for the micronucleus test. The dose level selected should ideally be the maximum tolerated dose level or that which produces some evidence of toxic ity up to a maximum recommended dose of 2000 mg/kg. The range

- finding toxicity test was also used to determine if the main test was to be performed using both sexes or males only. With no evidence of toxicity via the oral route, and following discussions with the Sponsor, it was considered necessary to

1 The study author reported doses of 0, 500, 1000, and 2000 mg/kg

- bw , which were calculated by adjusting for a purity of 50.5% (water content of 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

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27 investigate the intraperitoneal route of administration to maximi s e exposure of the test item.” [ Flanders 2013, p. 10]

In the range

- finding tests, groups of mice were dosed as follows: 1/sex at 1743 mg a. i./kg

- bw (oral) 1/sex at 872 mg a.i./kg

- bw (i.p.) 1/sex at 1308 mg a.i./kg

- bw (i.p.) 3 males and 1 female at 1743 mg a.i./kg

- bw (i.p.)

“All animals were dosed once only at the appropriate dose level [ using a dose volume of 10 mL/kg] by gavage using a meta l cannula or a hypodermic needle attached to a graduated syringe. The volume administered to each animal was calculated according to its bodyweight at the time of dosing.” [ Flanders 2013, p. 11]

“Animals were observed one hour after dosing and subsequently once daily for up to two days. Any deaths and evidence of overt toxicity were recorded at each observation. No necropsies were performed.” [ Flanders 2013, p. 11]

Main Test Procedure: M ale mice received a single dose of the test item via the intraperitoneal route at 436, 872, or 1743 mg a.i. /kg

- bw using a dose volume of 10 mL/kg

. Groups of 7 animals were dosed at 436 and 872 mg a.i./kg

- bw and 14 animals were dosed at 1743 mg a.i./kg

- bw. Se ven of the animals at the high dose were killed by cervical dislocation 24 hours following treatment and the remaining animals were killed after 48 hours.

“ Two additional groups of male mice were included in the study; one group (seven mice) was dosed v ia the intraperitoneal route with the vehicle alone (PBS) and a second group (five mice) was dosed orally with cyclophosphamide [at 50 mg/kg

- bw] . Cyclophosphamide is a positive control item known to produce micronuclei under the conditions of the test. T he vehicle and positive control group animals were killed 24 hours following dosing.” [ Flanders 2013, p. 11]

“ Animals were observed for signs of overt toxicity and death one hour after dosing and then once daily as applicable and immediately prior to te rmination.” [ Flanders 2013, p. 12]

Slide Preparation: “Immediately following termination ( i.e. 24 or 48 hours following dosing), both femurs were dissected from each surviving animal, aspirated with foetal bovine serum and bone marrow smears prepared following centrifugation and re

- suspension. The smears were air

- dried, fixed in absolute methanol, stained in May

- Grünwald/Giemsa, allowed to air

- dry and a cover slip applied using mounting medium.” [ Flanders 2013, p. 12]

Slide Evalution: “Stained bone marrow smears were coded and examined blind using light microscopy at x1000 magnification. Where possible, the incidence of micronucleated cells per 2000 polychromatic erythrocytes (PCE

- blue stained immature cells) per animal was scored. Micronuclei are normally circular in shape, although occasionally they may be oval or half

- moon shaped, andFinal HPV 2 Chemical Data Adequacy Review CAS RN 31138

- 65

- 5

28 have a sharp contour with even staining. In addition, the number of normochromatic erythrocytes (NCE

- pink stained mature cells) associated with 1000 erythrocytes was counted; these cells were also scored for incidence of micronuclei.” [ Flanders 2013, pg. 12]

“The ratio of polychromatic to normochromatic erythrocytes was calculated together with appropriate group mean values and standard deviations.” [ Flanders 2013, p. 13]

Criteria for a Positive Response : “A comparison was made between the number of micronu cleated polychromatic erythrocytes occurring in each of the test item groups and the number occurring in the vehicle control group.” [ Flanders 2013, p. 13]

“A positive mutagenic response would be demonstrated when a statistically significant, dose

respon sive, toxicologically relevant increase in the number of micronucleated polychromatic erythrocytes was observed for either the 24 or 48

- hour kill times when compared to the vehicle control group.” [ Flanders 2013, p. 13]

“If these criteria were not fulfil led, then the test item would be considered non

- genotoxic under these conditions of the test.” [ Flanders 2013, p. 13]

“A positive response for bone marrow toxicity would be demonstrated when the dose group mean polychromatic to normochromatic ratio was s hown to be statistically significantly lower than the vehicle control group.” [ Flanders 2013, p. 13]

RESULTS

Genotoxicity result: Negative

Cytotoxic concentration (if applicable) : > 17 43 mg a.i./kg

- bw (i.p.)

Results remarks :

Range

- Finding Study Results : No mortalities occurred during th e range

- finding study.

No clinical signs were observed i n animals dosed with the test item at 1743 mg a.i./kg

- bw via the oral route. “ Therefore, with no evidence of toxicity [via the oral route] the intraperito neal route of administration was investigated to maximise exposure of the test item.” [ Flanders 2013, p. 14]

Animals dosed intraperitoneally with the test substance at ≥ 872 mg a.i./kg

- bw showed clinical signs of hunched posture, ptosis, increased activity, and irritation and over

- grooming of the injection site (incidence data for each dose level were not reported) . Th is was considered to be evidence of modest toxicity . The dose levels chosen for the main test were 436, 872, and 1743 mg a.i./kg

- bw . Since there was no marked difference in the toxicity observed in male s and female s, the main test was performed using male mice only.Final HPV 2 Chemical Data Adequacy Review CAS RN 31138

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29 Main Test Results: No unscheduled deaths occurred in any of the dose groups in the main test. Clinical signs obser ved in animals dosed with the test item at ≥ 872 mg a.i./kg

- bw in both the 24 and 48

- hour dose groups included hunched posture and ptosis (incidence data for each dose level were not reported)

.
“Statistically significant decreases in the PCE/NCE ratio were not observed in any of the test item dose groups when compared to the vehicle control group. However, the observation of clinical signs was taken to indicate that systemic absorption had occur red and exposure to the target tissue had been achieved.” [ Flanders 2013, p. 15]

“There was no evidence of any statistically significant increases in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test item when comp ared to the vehicle control group.” [ Flanders 2013, p. 15]

“The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity o f cyclophosphamide under the conditions of the test.” [ Flanders 2013, p. 15]

CONCLUSIONS

“The test item was found not to produce a toxicologically significant increase in the frequency of micronuclei in polychromatic erythrocytes of mice under the conditions of the test.” [ Flanders 2013, p. 15]

STUDY RELIABILITY

[ 2 ] Reliable with restrictions. T he study report did not include incidence data on clinical signs observed in the main study (i.e., hunched posture and ptosis) or the range

- finding study (i.e., hunched posture, ptosis, increased activity, and irritation and over

- grooming o f the injection site) in animals dosed via the intraperitoneal route at 889 mg a.i./kg

- bw and above.

REFERENCE

Flanders, L. ( 2013 ) Sodium g lucoheptonate: Micronucleus test in the mouse

. Performing Laboratory: Harlan Laboratories Ltd.

, Shardlow, Derbyshire, UK. Project No. 41104690. Sponsor: Harcros Chemicals, Inc.

, Dalton, GA

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

- 65

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30 E

. DATA ADEQUACY SUMMARY TABLE

Endpoint Study Information Available Acceptable Data Gap

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

Toxicity to Aquatic Plants Yes Yes No Health Effects

Acute Toxicity Yes Yes No Repeated

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

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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 fish 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 from the guidelines are supplied, followed b y 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 Fish (Harris 2013) Relevant Guidelines: The study was conducted according to OECD TG 203

, Method C.1 of Commission Regulation (EC) No. 440/2008, and OPPTS 850.1075.

[A] Deviation 1: The pH of test solution preparations (i.e., solutions containing test substance and dilution water) in the definitive test was adjusted to 7 prior to the addition of test organisms. This pH adjustment altered the toxicity of the test solutions, as evidenced by the fact that toxicity (3/3 fish killed in extremis after 3 hours of exposure) was observed i n the range

- finding test at 889 mg a.i./L without pH adjustment, but no toxicity was observed in the definitive test at 889 mg a.i./L when the pH was adjusted to 7. If the test item causes a change in pH that increases the toxicity of the solution, then t he 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 (prepared by dechlorination and softening of tap water that had an average pH of 7.497) would be expected to increase the pH. If the resulting pH is outside of the physiologically acceptable range for freshwater fish of approximately 6 to 8 (per OPPTS 850.1075), then toxicity may result from the increase in pH caused by the addition of the t est 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 fish to these solutions, the testin g laboratory altered the toxicity of the test item to fish. T he results produced by the definitive study are not representative of the toxicity of the unaltered test substance. OECD TG 203, p. 3, Section “Description of the Method”, Sub

- section “Test solu tions”, item 14 states: “ The test should be carried out without adjustment of pH

. If there is evidence of marked change in the pH of the tank water after addition of the test substance, it is advisable that the test be repeated, adjusting the pH of the s tock solution to that of the tank water before addition of the test substance. This pH adjustment should be 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 caused. HCl and NaO H are preferred. ” OPPTS 850.1075, p. 1, Section (b ) “ Purpose” states: “ The purpose of the acute toxicity test with fish species is to help in the assessment of po ssible risk to similar species in natural environments, as an aid in determination of possible water quality criteria for regulatory purposes, and for use in correlation with acute testing of other species for comparative
purposes. ”Addendum to HPV2 Chemical Data Adequacy Review (Final ) CASRN 31138

- 65

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A

- 2 OPPTS 850.1075, p. 4, S ection (e) “Description of the method”, Sub

- section (3) “Solutions of test water”, item (vi) states: “The pH should not be adjusted after the addition of the test chemical or stock solution into dilution water.” Commission Regulation (EC) No. 440/2008, p. 592, Section C.1 “Acute Toxicity for Fish”, Sub

- section 1 “Method”, item 1.6.1 “Reagents”, sub

- item 1.6.1.1 “Solutions of test substances”, paragraph 5 states: “ The test should be carried out without adjustment of the pH

. If there is evidence of marke d change in the pH, it is advised that the test should be repeated with pH adjustment and the results reported. In that case, the pH value of the stock solution should be adjusted to the pH value of the dilution water unless there are specific

reasons not to do so. HCl and NaOH are preferred for this purpose. This pH adjustment should be made in such a way that the concentration of test substance in the stock solution is not changed to any significant extent

. Should any chemical reaction or physical pre cipitation of the test compound be caused by the adjustment, this should be reported. ”

S ummary of Quoted Material : The pH of test solutions in the definitive test should not be adjusted following the addition of the test substance. If the pH of the test solutions in the initial definitive test changes appreciably when 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 in itial definitive test. A purpose of the acute toxicity to fish test is to characterize the risk of the test substance to similar species in natural 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 fish test will not provide appropriate information for characterizing risk 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 : The analytical measurements of the test substance ranged from 57 to 184% of nominal concentrations. Test substance concentration measure ments for duplicate test solution samples varied greatly from one another; differences of up to 2.7

- fold were observed between duplicate samples, suggesting that analytical techniques for measuring test substance concentrations may not have been reliable a nd/or accurate . Measured test substance concentrations of “old” test solutions (test medium to which fish had been exposed for 24 hours, sampled just prior to solution renewal at 24, 48, 72, and 96 hours) were up to 105% different from concentrations meas ured in “fresh” test solutions. No explanation was provided for the variability in measured test substance concentrations. Given the large differences between measured and nominal concentrations , the endpoint values (LC 50 , LOEC, and NOEC ) should be based on the mean measured, rather than nominal, concentrations. OECD TG 203, p. 1, Section “Information on the Test Substance”, item 4 states: “... A reliable analytical method for the quantification of the substance in the test solutions must also be available. ” OECD TG 203, pp. 1

- 2, Section “ Validity of the Test ”, item 6 states: “For a test to be valid the following conditions should be fulfilled:”; and t he fourth condition states: “ there

must be evidence that the concentration of the substance being tested has been satisfactorily maintained , and preferably it should be at least 80 per cent of the nominal concentration throughout the test. If the deviation from the nominal concentration is greater than 20 per cent, results should be based on the measured concentration. ”Addendum to HPV2 Chemical Data Adequacy Review (Final ) CASRN 31138

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A

- 3 OPPTS 850.1075, p. 2 , Section ( c ) “ Principle of the test ”, item ( 3 ) “Information on the test substance” states: “... A reliable analytical method for quantification of test substance concentrations must be available. ” OPPTS 850.1075, p. 2 , Section ( d ) “ Validity of the test ”, item ( 4 ) states: “ Measured concentrations are required if the test chemical is unstable or a flow

- through diluter system is employed. Exception may be made in cas es where hydrolysis studies indicate chemical to be stable (< 5 percent degradation) in 96 h at a pH comparable to test dilution water. In any case there must be evidence that test concentrations remained at least 80 percent of the nominal concentrations throughout the test or that mean measured concentrations are an accurate representation of exposure levels maintained throughout the test period. ” OPPTS 850.1075, p. 8 , Section ( f ) “ Performance of the test ”, Sub

- section ( 2 ) “Test concentrations”, item (ii i) states: “ No more than 25 percent variation is allowed between test concentrations within the same treatment during the test. ” Commission Regulation (EC) No. 440/2008, p. 591, Section C.1 “Acute Toxicity for Fish”, Sub

- section 1 “Method”, item 1.5 “Quality Criteria”, paragraph 4 states: “The concentrations of the test substance shall be maintained to within 80% of the initial concentrations throughout the duration of th e test.”; and paragraph 5 states: For substances which dissolve easily in the test medium, yielding stable solutions..., the initial concentration can be taken as being equivalent to the nominal concentration. Evidence shall be presented that the concentrat ions have been maintained throughout the test and that the quality criteria have been satisfied.”; and p aragraph 6 states: “For substances that are (i) poorly soluble in the test medium, or (ii) capable of forming stable emulsions or dispersions, or (iii) not stable in aqueous solutions, the initial concentration shall be taken as the concentration measured in solution... at the start of the test.”; and paragraph 7 states: “In any of these cases, further

measurements must be made during the test to confirm the actual exposure concentrations or that the quality criteria have been met.” Commission Regulation (EC) No. 440/2008, p. 595, Section C.1 “Acute Toxicity for Fish”, Sub

- section 2 “Data and Evaluation”, paragraph 5 states: “If it is observed that the stability or homogeneity of the test substance cannot be maintained, this should be reported and care should be taken in the interpretation of the results.”

Summary of Quoted Material : Measured concentrations of test solutions throughout the test shall be within 80% of initial measured concentrations. Variation among measured concentrations for a given treatment level is not allowed to exceed 25%. Evidence must be provided that test concentrations were maintained throughout th e test. Measured concentrations should be within 80% of nominal concentrations. If test concentrations deviate from nominal concentrations by more than 20%, the results should be based on measured concentrations. If the study results indicate that the s tability or homogeneity of the test substance cannot be maintained, the study may not be acceptable or the study should be interpreted with caution because the results may not be reproducible. Interpretation: Test concentrations must be determined by a r eliable analytical method that provides an accurate representation of exposure levels. If the results (i.e., endpoint values) are based upon nominal concentrations, measured test concentrations must be within 80% of nominal concentrations. If measured co ncentrations for a given treatment level vary by more than 25%, the test is not considered acceptable.Addendum to HPV2 Chemical Data Adequacy Review (Final ) CASRN 31138

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A

- 4 REFERENCE

Harris, S. (2013) Sodium glucoheptonate: Acute toxicity to rainbow trout ( Oncorhynchus mykiss )

. Performing Laboratory: Harlan Laboratories Ltd., Shardlow, Derbyshire, UK. Study No. 41104694. Sponsor: Harcros Chemicals, Inc., Dalton, GA. Study report available online at http://www .regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0715 as of January 14, 2014

.