SRC TR

- 13

- 045

HPV 2 TEST RULE DATA ADEQUACY REVIEW : JUNE 2013 UPDATE ( FINAL )

D

- gluco

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

- , CASRN 31138

- 65

- 5

Prepared by:

Megan Nelson, Amy Nguyen, 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 FG007.3.009.001

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

June 13 , 2013Final HPV 2 Chemical Data Adequacy Review (June 2013) 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

................................ ................................ ........... 30Final HPV 2 Chemical Data Adequacy Review (June 2013) 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 June 13, 2013 ).

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 June 13, 2013 ) 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 June 13, 2013 ) 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 June 13, 2013 ) 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

June 13, 2013) 2
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 June 13, 2013 )

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 June 13, 2013.

2 Harcros Chemical, Inc. su bmitted 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 (June 2013) CAS RN 31138

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- 5

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 availab le 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 document 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 summarizing data avai lability/adequacy for each SIDS endpoint is included on the last page of this document (Section E).

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

Data 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 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 a djustment, but no toxicity was observed in the definitive test at 889 mg a.i./L when pH was adjusted to 7. Any toxicity resulting from a change in pH caused by the test substance is considered to be essential to understanding the toxic effects of the test substance. The results produced by the definitive study are not representative of the toxicity of the
unaltered test substance.

(2) The test item was boiled prior to preparation of test solutions. According to the study author, boiling was intended to remove any crystals that had formed. However, boiling the test substance may have caused the test substance to react and form other chemical substances. Because the chemical composition of the boiled test substance is unknown, it
is unclear whether the t est organisms were exposed to CASRN 31138

- 65

- 5 or a different
chemical substance. 1

1 Although the study report included a report describing analytical measurements of test substance concentrations (Appendix 2: Analytical Investigations), this report is incomplete. In order to determine whether or not the chemical identity of the test su bstance was changed upon boiling, the submitter would need to provide the mass spectrum that corresponds to the peak shown in the chromatogram.Final HPV 2 Chemical Data Adequacy Review (June 2013) CAS RN 31138

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3 (3) The analytical measurements of the test substance ranged from 57 to 184% of nominal concentrations. Given the large variation in measurements, the endpoint values (LC 50 , LOEC, and NOEC ) should be based on the mean measured, rather than nominal,
concentrations.

Therefore, the submitter needs to repeat the acute toxicity to fish test, implementing the correct procedures to avoid the deficiencies identified above.

Data h ave not yet been submitted for the following toxicity testing requirements : acute toxicity

to Daphnia study (component of C1 required testing) and combined repeated

- dose toxicity study with the reproduction/developmental toxicity screening test (F1 testing) . A study plan for these endpoints was submitted by Harcros Chemicals, Inc. on July 29, 2011 (available online at http://www.regulations.gov/#!documen tDetail;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0639 as of June
13 , 2013).Final HPV 2 Chemical Data Adequacy Review (June 2013) CAS RN 31138

- 65

- 5

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 pH was adjusted to 7. Any toxicity resulting from a change in pH caused by the t est substance is considered to be essential to understanding the toxic effects of the test substance. The results produced by the definitive study are not representative of the toxicity of the
unaltered test substance. (2) The test item was boiled prior to preparation of test solutions. According to the study author, boiling was intended to remove any crystals that had formed. However, boiling the test substance may have caused the test substance to react and form other chemical
substances. Because the ch emical composition of the boiled test substance is unknown, it is unclear whether the test organisms were exposed to CASRN 31138

- 65

- 5 or a different
chemical substance. 1 (3) The analytical measurements of the test substance ranged from 57 to 184% of nominal concentrations. Given the large variation in measurements, the endpoint values (LC 50 , 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 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 follows:

1 Although the study report included a report describing analytical measurements of test substance concentration s (Appendix 2: Analytical Investigations), this report is incomplete. In order to determine whether or not the chemical identity of the test substance was changed upon boiling, the submitter would need to provide the mass spectrum that corresponds to the peak shown in the chromatogram.Final HPV 2 Chemical Data Adequacy Review (June 2013) CAS RN 31138

- 65

- 5

5 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/#!docu mentDetail;D=EPA

- HQ

OPPT

- 2007

- 0531

- 0639 as of June 10 , 2013 . 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

.

Analytical monitoring: Yes, only at the highest concentration ( 889 mg a

. i

. /L) in the definitive

test

Exposure duration: 96 hours

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. 3

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

- 5

3 The study author erroneously reported 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 (June 2013) CAS RN 31138

- 65

- 5

6 Test method/conditions remarks: Test water: “Laboratory tap water 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 qu antitation, 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 ranged from 0.000 to 0.091 mg Cl 2 /L.” [Harris 2013, p. 11]

Range

- Finding Test Procedure: Two range

- finding tests were co nducted 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 analysis.” [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]

Definitive Test Procedure:
Based on the results of the r ange

- finding tests

, the concentrations used in the definitive test were 0, 89, 160, 284, 498, and 889 mg a.i./L.Final HPV 2 Chemical Data Adequacy Review (June 2013) CAS RN 31138

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7 “ 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. ” [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 placed 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 dawn 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 tes t 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 re corded 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 preparations

. 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 therm ometer. The temperature was also monitored in the control hourly using a

Testo temperature logger. ” [Harris 2013, p. 13]

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. ” [H arris 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.Final HPV 2 Chemical Data Adequacy Review (June 2013) CAS RN 31138

- 65

- 5

8 RESULTS

Endpoint value(s): 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 was not adjusted to within specification. Adjustments to pH were made during the definitive test and hence it was consi dered 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

... rec orded in the control vessel approximat ely every hour using a Testo temperature logger

... ranged from 12 ° C to 14 ° C.” 2 [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

1 While the study author considers the pH effect to not be a “true” toxic effect, EPA disagrees. Any toxicity resulting from a c hange in pH caused by the test substance is considered by EPA to be a true toxic effect.

2 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 (June 2013) CAS RN 31138

- 65

- 5

9 impact on the outcome o r integrity of the test as no adverse effects were observed in the control

grou p.” [Harris 2013, p. 15]

Verification of Test Concentrations: “Analysis of the ... [ second set of duplicate samples taken for analysis of test substance concentration] 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 preparation at 48 hours was measured to be 184% of nominal, however, as the corr esponding 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 samples.

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 t oxicity was observed in the definitive test at 889 mg a.i./L when pH was adjusted to 7. Any toxicity resulting from a change in pH caused by the test substance is
consi dered to be essential to understanding the toxic effects of the test substance. The results produced by the definitive study are not representative of the toxicity of the
unaltered test substance. (2) The test item was boiled prior to preparation of test so lutions. According to the study author, boiling was intended to remove any crystals that had formed. However, boiling the test substance may have caused the test substance to react and form other chemical
substances. Because the chemical composition of the boiled test substance is unknown, it is unclear whether the test organisms were exposed to CASRN 31138

- 65

- 5 or a different
chemical substance. 1
(3) The analytical measurements of the test substance ranged from 57 to 184% of nominal
concentrations. Given t he large variation in measurements, the endpoint values ( LC 50 ,

1 Although the study report included a report describing analytical measurements of test substance concentrations (Appendix 2: Analytical Investigations), this report is incomplete. In order to determine whether or not the chemical identity of the test su bstance was changed upon boiling, the submitter would need to provide the mass spectrum that corresponds to the peak shown in the chromatogram.Final HPV 2 Chemical Data Adequacy Review (June 2013) CAS RN 31138

- 65

- 5

10 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 (June 2013) CAS RN 31138

- 65

- 5

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 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
103 8 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 June 10 , 2013 . 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 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 ingredie nt (a.i.) = CASRN 31138

- 65

- 5Final HPV 2 Chemical Data Adequacy Review (June 2013) 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)

Exposur e 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 con trol and test concentration. The 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

conce ntrations 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 (June 2013) CAS RN 31138

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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 illu mination (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) o f each solution was 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 nomina l 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 correcte d for 44.02% purity.Final HPV 2 Chemical Data Adequacy Review (June 2013) CAS RN 31138

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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 wit h 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 following 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
controls 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 (June 2013) CAS RN 31138

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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 resu lts 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 inhibitio n 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 (June 2013) CAS RN 31138

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16 were significant (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 m g 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 mg 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, and 889 mg a.i./L, respectively; effects on biomass were significant (P < 0.5) at conce ntrations ≥ 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, 89, 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 the test began, the pH of test solutions was 7.7, 7.2, 7.4, 8.7, 9.3, and 9.6
at concentratio ns 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 (June 2013) CAS RN 31138

- 65

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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 t est 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 unadjusted, was similar to the tox icity 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 (June 2013) CAS RN 31138

- 65

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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 June 10 , 2013 . Based on this information, the purity of the test
substance was 44.02% a.i. 2 (23.4% beta sodium gluco heptonate + 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.nih.gov/chemidplus/ ); however, based on the chemical name prov ided (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 (June 2013) 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 d osing. Treatment of animals was sequential. Sufficient 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, 1778, 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 obse rved 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 after
treatment.” [ Sande rs 2013, p. 9]

1 The study author erroneously reported equivalent 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 (June 2013) 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 abdominal and thoracic cavit ies 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 were noted.” [ Sand ers 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 the 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 (June 2013) 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 Mar ch 5, 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 A mmonia
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 June 10, 2013. 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 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 (June 2013) 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 formulation 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 pe rformed: 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 live r 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 substance. 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, 500, 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 aut hor 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 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 (June 2013) CAS RN 31138

- 65

- 5

23 I n experiment 1, concentrations of 44, 131, 436, 1308, and 4358 μg a.i./plate 1 were assayed in trip licate 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 were 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

- nitrosogu anidine (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

- relate d 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 repo rted when an experiment 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 tes t 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 ite m, 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 above have been corrected for 44.02% purity.Final HPV 2 Chemical Data Adequacy Review (June 2013) 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, conf irming 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 (gu ideline 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 Escherichia 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 Chem ical, 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 (June 2013) 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

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- 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 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
103 8 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 June 10 , 2013 . 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 Japanese 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 provide d (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 (June 2013) 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 prepared 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
ad ministration for the micronucleus test. The dose level selected should ideally be the maximum tolerated dose level or that which produces some evidence of toxicity up to a maximum recommended dose of 2000 mg/kg. The range

- finding toxicity test was also u sed 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 (June 2013) CAS RN 31138

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27 investigate the intraperitoneal route of admi nistration 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 an d 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 metal 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 afte r 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 tes t 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. Seven of the animals at the high dose we re 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 via 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. The 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 termination.” [ Flanders 2013, p. 12]

S lide 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

- suspensi on. 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. Mi cronuclei are normally circular in shape, although occasionally they may be oval or half

- moon shaped, andFinal HPV 2 Chemical Data Adequacy Review (June 2013) CAS RN 31138

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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 micronucleated 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

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

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

“If these criteria were not fulfilled, 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 shown 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 intraperitoneal 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 (June 2013) 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
observed 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 co ntrol group. However, the observation of clinical signs was taken to indicate that systemic absorption had occurred 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 compared 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 of cyclophosphamide under the conditions of the test.” [ Flanders 2013, p. 15]

CONC LUSIONS

“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 (June 2013) CAS RN 31138

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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 No

– Yes

Toxicity to Aquatic Plants Yes Yes No
Health Effects

Acute Toxicity Yes Yes No Repeated

- Dose Toxicity No

– Yes
Gene Mutation Yes Yes No
Chromosomal Aberrations Yes Yes No Reproductive Toxicity No

– Yes Developmental Toxicity No

– Yes