SRC TR

- 13

- 052

HPV2 TEST RULE DATA ADEQUACY REVIEW ( FINAL ) : ACUTE TOXICITY TO FI SH, ACUTE TOXICITY T O DAPHNIA , TOXICITY TO ALGAE,

AND CHROMOSOMAL ABERRATI ONS

Ethanedioic Acid, CASRN 144

- 62

- 7

Prepared by:

Stephen Bosch, Lisa Ingerman, William Richards, and Teresa Manyin

Defense and Environmental Solutions Division SRC, Inc.
7502 Round Pond Road North Syracuse, NY 13212

Contract No. EP

- W

- 09

- 027
Task FG007.3 .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

July 26 , 2013Final HPV 2 Chemical Data Adequacy Revie w CASRN 144

- 62

- 7

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

................................ ................................ .............................. 3

D

- 1. Acute Toxicity to Fish

................................ ................................ ................................ ....... 3

D

- 2. Acute Toxicity to Aquatic Invertebrates

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

D

- 3. Toxicity to Aquatic Plants

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

D

- 4. Genetic Toxicity

– Chrom osomal Aberrations

................................ ................................ 27Final HPV 2 Chemical Data Adequacy Revie w CASRN 144

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- 7

1 A. SOURCE MATERIAL USED FOR ASSESSMENT

This document evaluates a submission (dated March 27 , 2013 ) from Greenwich Chemical Consulting, Inc. on behalf of Univar USA Inc. and WEGO Chemical and Mineral Corporation that includes data from toxicity testing of ethanedioic acid, hydrate (1:2) (CASRN 6153

- 56

- 6), which is the hydrated form of ethanedioic acid (CASRN 144

- 62

- 7) . These data were received by EPA in response to the final test rule, “Testing of Cer tain 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.g ov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0579 as of July 23, 2013).

The HPV2 test rule requires the following toxicological testing for CASRN 144

- 62

- 7: C1 (consisting of acute toxicity to fish, acute toxicity to Daphnia , and toxicity to algae testi ng if log

K
ow < 4.2 or chronic toxicity to Daphnia and toxicity to algae testing if log K
ow ≥ 4.2) and E2 (chromosomal aberration or micronucleus test)

. Given that the log K
ow of CASRN 144

- 62

- 7 has been reported to be less than zero 1

, the C 1 aquatic toxic ity testing requirement is considered to
include acute toxicity to fish, acute toxicity to Daphnia , and toxicity to algae testing.

In its March 27, 2013 submission (available online at http://www.regulations.gov/#!document Detail;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0712 as of July 23, 2013), Greenwich Chemical Consulting, Inc. supplied data on behalf of Univar USA Inc. and WEGO Chemical and Mineral Corporation from the followi ng toxicity tests, conducted with CASRN 6153

- 56

- 6 :

• Acute toxicity test in zebrafish

• Acute immobilization test in Daphnia magna

• Growth inhibition test in Pseudokirchneriella subcapitata

• In vitro chromosomal aberration test in Chinese hamster V79 lung cell s

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

Although the test rule applies to the anhydrous form of ethanedioic acid (CASRN 144

- 62

- 7), t he submitted tests were conducted using th e dihydrate form of ethanedioic acid (CASRN 6153

- 56

- 6). The sponsors, Univar USA Inc. and WEGO Chemical and Mineral Corporation, argued that testing of ethanedioic acid in its anhydrous form would be impractical because ethanedioic acid is highly hygrosco pic, meaning that any exposure of the anhydrous form to water (including water present in the air) would quickly result in the formation of the dihydrate material due to the absorption of water. Additionally, the sponsors indicated that the dihydrate is t he commercially
available form of ethanedioic acid and would be the relevant form for human exposure and
environmental release. 2 EPA finds the use of the dihydrate form of ethanedioic acid (CASRN
6153

- 56

- 6) as a test substance acceptable for conducting to xicity tests required for CASRN 144

- 62

- 7 in the HPV2 test rule.

1 Two values,

- 0.81 and

- 0.43, are reported by Verschueren (2001), Han dbook o f Environmental Data on Organic Chemicals, 4 th edition, New York: John Wiley & Sons, Vol. 2, p. 1691.

2 See cover letter of March 27, 2013 submission (available online at http://www.regulations.gov/#!document Detail;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0712 as of July 23, 2013).Final HPV 2 Chemical Data Adequacy Revie w CASRN 144

- 62

- 7

2 B. EVALUATING DATA ADEQUACY

The submitted data are evaluated for adequacy in Section D. Each study was summarized according to a standard format and assigned a Klimisch reliability code ba sed 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 req uirements. 1 If the data available for the endpoint were determined to be inadequate, a basis for that conclusion is supplied.

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

Data submitted by Greenwich Chemical Consulting, Inc., on beh alf of Univar USA Inc. and WEGO Chemical and Mineral Corporation, on March 27, 2013 are adequate to satisfy the C1 (acute toxicity to fish, acute toxicity to Daphnia , and toxicity to algae) and E2 (chromosomal aberrations) testing requirements for CASRN 14 4

- 62

- 7 promulgated in the final HPV2 test rule.

There are no outstanding test rule requirements for toxicity testing of CASRN 144

- 62

- 7.

1 T he HPV 2 test rule specifies that acute fish and daphnid toxicity testing be conducted according to ASTM E729

- 96, algae toxicity testing be con ducted according to ASTM guideline E 1218

- 04 ε1 , and in vitro chromosomal aberration testing be conducted according to 40 CFR 799.9537. However , EPA previously granted a request from
the sponsors to follow OECD test guidelines instead (request granted via email from P. Campanella (EPA) to J. Massing ale (Greenwich Chemical Consulting) on January 10, 2012 and February 23, 2012 ) , as indicated in the cover letter of the sponsors’ March 27, 2013 submission (available online at http://www.regulations.gov/#!document Detail;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0712 as of July 23, 2013).Final HPV 2 Chemical Data Adequacy Revie w CASRN 144

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- 7

3 D . DATA FOR SIDS ENDPOINTS

D

- 1

. Acute Toxicity to Fish

Summary of endpoint: Adequate

Study 1: Acute toxicity study in z ebrafish (CiToxLAB Hungary Ltd., 2013a)

Title: Acute toxicity test with ethanedioic acid, hydrate (1:2) [CASRN 6153

- 56

- 6] (hydrated form of ethanedioic acid [CASRN 144

- 62

- 7]) on zebrafish ( Brachydanio rerio )

This study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Ethanedioic acid, hydrate (1:2), CASRN 6153

- 56

- 6

Remarks: Batch No. YP1

- 20120528C. Ethanedioic acid (CASRN 144

- 62

- 7, aka oxalic acid) was tested in its hydrated (dihydrate) form , as white crystals that were 99.69% pure and supplied by the study sponsor

. 1

Conversion factor for calculating mg of ethanedioic acid (C 2 H 2 O 4 ) from mg of the dihydrate (C 2 H 2 O 4 ·2H 2 O) = 0.71 [ratio of molecular weight of ethanedioic acid (90.03 g/mol) to

e thanedioic acid dihydrate (126.07 g/mol)].

METHOD

Method/guideline followed: OECD TG 203 , “ Fish, Acute Toxicity Test ” (1992); Commission Regulation (EC) No 440/2008 , C.1, “Acute Toxicity for Fish” (2008); OPPTS 850. 1075, “ Fish Acute Toxicity Test, Fresh water and Marine” (1996)

Test type: Acute toxicity to fish

- static conditions

GLP compliant?: Yes

Year study was performed: 201 2

Test species: Zebrafish ( Brachydanio rerio ) ; source: “Black Molly” Fish Farm; Ágnes Németh
(self

- employed), Veszp rém, Hungary ; body length: 2.6

- 3.4 cm in the definitive study and 2.7

-

1 The certificate of analysis (COA) provided in the study report identifies the test substance as oxalic acid (synonymous with ethanedioic acid), lot no. YP1

- 20120528C, but does not state whether the substance is in the anhydrous or dihydrate form. Furthermore, the composition provided in the COA does not list water as a component of the test substance, which suggests that the test substance was a nhydrous. Therefore, although the test substance is identified as the dihydrate form of ethanedioic acid throughout the text of the study report, the COA does not confirm this identity of the test substance.Final HPV 2 Chemical Data Adequacy Revie w CASRN 144

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4 3.3 cm in the complementary study; average body weight: 0.30 g in the definitive study and 0.36 g in the complementary study

Analytical monitoring: Yes

Exposure duration: 96 hour s

Concentrations a tested: Concentration range

- finding test (unadjusted pH) : Nominal: 0, 0.1, 1, 10

, and 100 mg/L Definitive test (Phase 1 , unadjusted pH ): Nominal: 0, 6.25, 12.5, 25, 50, and 100 mg/L
Geometric mean measured b : 0, 4.48, 6.77, 14.62, a nd 34.22 for the nominal concentrations of 0, 6.25, 12.5, 25, and 50 mg/L, respectively; for the nominal concentration of 100 mg/L, a measured value (96.6 mg/L) was reported only for samples analyzed at the start of the study because all fish had died by 3 hours on test. Complementary test (Phase 2, adjusted pH): Nominal: 0, 50, and 100 mg/L
Geometric mean measured b : 0, 15.99, and 32.94 mg/L

a Concentrations are expressed as mg/L of ethanedioic acid, hydrate (1:2).

b Geometric means of initial and 96

- hour concentrations.

Test method/conditions remarks: Preliminary R ange

- finding T est (unadjusted pH) : Test substance concentrations in the definitive test were based on a preliminary test in which groups of 4 fish were exposed to ethanedioic acid, hyd rate (1:2) at nominal concentrations of 0, 0.1, 1, 10

, or 100 mg/L under static conditions for
96 hours.

Definitive Test (Phase 1, unadjusted pH) : Groups of 7 fish were exposed to nominal concentrations of 0, 6.25, 12.5, 25, 50

, or 100 mg/L under stati c conditions for 96 hours . At the highest test concentration, the study was terminated after 3 hours because all fish had died

. Except for the highest concentration tested (for which only the initial concentration was measured), c oncentrations were measu red at the beginning and end of testing.

Complementary Test (Phase 2, adjusted pH) : Groups of 7 fish were exposed to nominal concentrations of 0, 50

, or 100 mg/L under static conditions for 96 hours. Concentrations were measured at the beginning and end of testing.

The complementary test was conducted because pH values were below 6 in test solution s at the two highest concentrations in the definitive (Phase 1) test. In particular, “Because there was evidence of marked change in the pH of the test water after addition of the test substance, and the pH was below 6 in the definitive test at the two highest concentration levels of 50 and 100 mg/L, a complementary study was carried out, adjusting the pH of the stock solution to that of the
dilution water bef ore addition of the test substance. The pH was adjusted through the addition of

NaOH.” [CiToxLAB 2013a, p. 14] The complementary test therefore tested only 50 and 100Final HPV 2 Chemical Data Adequacy Revie w CASRN 144

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- 7

5 mg/L nominal concentrations at a pH within the range (6

- 8.5) recommended by OECD TG 20 3. (After adjustment, the pH of test solutions at nominal concentrations of 50 and 100 mg/L was ~7.4 at initiation of exposure)

.

Test solutions : Reconstituted water (ISO medium, per OECD TG 20 3 ) was used as dilution water for the range

- finding, defini tive

, and complementary tests

. Reconstituted water was used rather than filtered tap water as originally planned for the study “ due to the problem with analytical measurement of the test substance in aquarium water (based on the analytical method developm ent and validation).” [CiToxLAB 2013a, p. 17] ISO medium had an approximate theoretical total hardness of 249 mg CaCO 3 /L.

A stock solution of 100 mg/L was prepared and test solutions were prepared by dilution of the
stock solution in ISO medium.

“In th e complementary experiment the pH of the stock solution was adjusted and then the appropriate dilution was prepared and distributed into test vessels prior to introduction of fish.” [CiToxLAB 2013a, p. 11]

Test C onditions : Static conditions were used “si nce the test material was found to be stable in water over the test period...” [CiToxLAB 2013a, p. 8] during the analytical method validation [see CiToxLAB 2013b, pp. 13

- 14]. The validity of this statement is not supported by the analytical measurements con ducted on samples from the definitive and complementary tests. In particular, the problem with maintaining test concentrations throughout the study suggests that a
semi

- static or flow

- through system should have been utilized.

For the definitive and com plementary tests, one aquarium was used for each test concentration; each aquarium contained 7 fish and 5 L of test solution. The photoperiod was 16 hours light:8 hours dark. During the test, the fish were not fed and loading (g fish/L test solution) at experiment initiation was 0.31

- 0.49 for the definitive test and 0.48

- 0.52 for the complementary test. Before use in the test, the fish were acclimatized in ISO medium for 5 days (preliminary test) or at least 12 days (definitive and complementary tests).

Observations : “ The observations of fish were carried out in the following intervals: at 3, 6, 24, 48, 72 and 96 hours. Mortality and any sub

- lethal effect were determined in each observation period. The test conditions (pH, temperature, oxygen satu ration) were measured daily during the

test. The body weights of the fish were recorded before the introduction of fish. The body weight of 7 fish per aquarium was registered. The loading of the aquaria was calculated on the basis of these body weights (g fish/litre testing liquid).” [CiToxLAB 2013a, p. 14]

Analytical M onitoring : Concentrations of ethanedioic acid, hydrate (1:2) were analytically determined using a validated HPLC

- UV method that is summarized in CiToxLAB 2013a (Appendix 4) and detailed in CiToxLAB 2013b. Samples (number not indicated) were taken for analysis at the beginning and end of the definitive and complementary tests.Final HPV 2 Chemical Data Adequacy Revie w CASRN 144

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6 Statistical E valuation : EC 50 values (based on measured concentrations), along with 95% confidence limits, were calculated by Probit analysis using SPSS PC+ software

. The LOEC and NOEC values were determined directly from the raw data.

RESULTS

Endpoint value(s): E xcept for the highest test concentration, for which concentration was analytically measured only at the start of the study, all endpoint values are based on geometric mean measured concentrations (means of initial and 96

- hour concentrations).

Based on t esting with unadjusted pH (definitive test ) :

Ethanedioic acid, hydrate (1:2) , CASRN 6153

- 56

- 6 : 96

- hour LC 50 = 56.0 mg/L; 95% confidence interval = 35.83

- 105.46 mg/L

96

- hour LOEC (mortality or signs of toxicity) = 96.60 mg/L

96

- hour NOEC (mortality or signs of toxicity) = 34.22 mg/L

Ethanedioic acid , CASRN 144

- 62

- 7 ; c orresponding equivalent toxicity values were calculated
from the values for e thanedioic acid, hydrate (1:2) using a conversion factor of 0.71 (ratio of molecular weight of the acid to the dihydrate):

96

- hour LC 50 = 39. 8 mg/L; 95% confidence interval = 25.4

- 74. 9 mg/L

96

- hour LOEC (mort ality or signs of toxicity) = 68. 6 mg/L

96

- hour NOEC (mortality or signs of toxicity) = 24.3 mg/L

Based on testing with adjusted pH (complementary test ) :

Ethanedioic acid, hydrate (1:2) , CASRN 6153

- 56

- 6:
96

- hour LC 50 > 32.94 mg/L

96

- hour NOEC (mortality or signs of toxicity) = 3 2

. 94 mg/L (highest concentration tested )

Ethanedioic acid , CASRN 144

- 62

- 7; equivalent toxicity values:

96

- hour LC 50 > 23.4 mg/L

96

- hour NOEC (mortality or signs of toxicity) = 23

. 4 mg/L (highest concentration tested )

Results rema rks:
Measured Test Concentrations : F or the definitive test, n ominal and mean measured
concentrations

, with percentage of nominal values

, are shown in the following table. For nominal concentrations up to 50 mg/L, measured concentrations declined 41

- 76% from the initiation of the exposure to the end of exposure. No measured concentration wa s reported at 96
hours for the highest test concentration (100 mg/L nominal) because all fish had died by 3 hours

on test

.Final HPV 2 Chemical Data Adequacy Revie w CASRN 144

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- 7

7 Measured concentrations of ethanedioic a cid, hydrate (1:2) with 95% confidence intervals
in the definitive test

Nominal
Concentration (mg/L) Measured Concentration at Start of Exposure Measured Concentration at End of Exposure

Concentration (mg/L) Percentage of Nominal (%) Concentration (mg /L) Percentage of Nominal (%)

0 Not detected

- Not detected

- 6.25 7.32 ± 0.24 117 2.74 ± 0.27 44

12.5 13.9 ± 0.30 111 3.30 ± 0.22 26

25 26.2 ± 0.47 105 8.16 ± 2.70 33

50 44.7 ± 4.47 89 26.2 ± 5.39 52

100 96.6 ± 0.25 1 97

- - 1 A t the nominal concent ration of 100 mg/L , a measured concentration was determined only at the start of exposure because all fish at that concentration died within the first 3 hours of

exposure.

For nominal concentrations of 0, 6.25, 12.5, 25, and 50 mg/L, geometric mean measur ed
concentrations (means of initial and 96

- hour values) were 0, 4.48, 6.77, 14.62, and 34.22 mg/L, respectively. The rationale for determining geometric mean values was: “As the measured concentrations deviated more than 20 per cent from the nominal in mo st cases, biological results are based on the measured geometric mean concentrations.” [CiToxLAB 2013a, p. 13]. The geometric mean measured concentrations appear to have been calculated using start

- and end

- of

study concentrations.

For the complementar y test , geometric mean measured concentrations were 15.99 and 32.94 mg/L for nominal concentrations of 50 and 100 mg/L, respectively. Although concentrations were measured at the beginning and end of testing, measured concentrations at each of these time points were not reported (unlike the definitive test).

Results of the Preliminary Range

- Finding Test (unadjusted and unmeasured pH) : Fish mortality was 100% at 100 mg/L and 0% at ≤ 10 mg/L. Based on these data, five test concentrations ranging from 6.25 to 100 mg/L in a geometric series (separation factor of 2) and one untreated control were selected for the definitive test. Test substance concentrations and pH were not measured in the range

- finding test.

Results of the Definitive Test (with unadjusted pH) : Incidences of fish mortality and pH values observed in the definitive test are shown in the following table.Final HPV 2 Chemical Data Adequacy Revie w CASRN 144

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- 7

8 Cumulative mortality and pH values in the definitive test

Nominal
Concentration (mg/L) Geometric Mean
Measured
Concentration (mg/L) Cum ulative Mortality at 96

Hours pH 1

0 h 24 h 48 h 72 h 96 h

0 0 0/7 7.70 7.69 7.62 7.61 7.79

6.25 4.48 0/7 7.29 7.76 7.65 7.55 7.62

12.5 6.77 0/7 7.06 7.71 7.60 7.50 7.65

25 14.62 0/7 6.61 7.34 7.47 7.39 7.60

50 34.22 0/7 4.93 5.36 5.81 7.29 7.45

1 00 96.6 7/7 3.28

- - - - 1 The pH at the nominal concentration of 100 mg/L was only measured at 0 hours because all fish at that concentration died within the first 3 hours of exposure.

The incidence of mortality was 7/7 at 100 mg/L vs. 0/7 at all concen trations ≤ 50 mg/L, including the control. No sublethal effects were observed. The pH of test water decreased with increasing test substance concentration and the pH at test initiation was below 6 at the two highest nominal concentrations of 50 mg/L (pH 4.93) and 100 mg/L (pH 3.28). During the definitive test, water temperature ranged from 20.0 to 20.8° C and dissolved oxygen from 90 to
99% of air saturation value.

Results of the Complementary Test (with adjusted pH) : Incidences of fish mortality and pH values observed in the complementary test are shown in the following table.

Cumulative mortality and mean pH values in the complementary test

Nominal
Concentration (mg/L) Geometric Mean
Measured
Concentration (mg/L) Cumulative Mortality at 96

Hours pH

0 h 24 h 48 h 72 h 96 h

0 0 0/7 7.43 7.45 7.42 7.43 7.42

50 15.99 0/7 7.43 7.44 7.45 7.44 7.43

100 32.94 0/7 7.00 7.53 7.55 7.51 7.50

OECD TG 203 indicates that the acute test in fish should initially be “carried out without adjustment of pH.” O ECD TG 203 further indicates 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 stock solution to that of the tank water before additio n of the test substance.” Because pH values fell below 6 for the two highest test substance concentrations of the definitive test, the complementary test was conducted by retesting the 50

- and 100

- mg/L concentrations with the pH of stock solutions adjuste d to the pH of the dilution water prior to exposure of the fish to the test solutions. Initial pH values at 50 and 100 mg/L were 7.43 and 7.00, respectively, compared with 7.43 at 0 mg/L. No mortality or sublethal effects wereFinal HPV 2 Chemical Data Adequacy Revie w CASRN 144

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- 7

9 observed in this test. Dur ing the complementary test, water temperature ranged from 20.2 to 20.7°C and dissolved oxygen from 94 to 98% of air saturation value.

Selection of Data for Derivation of the LC 50 : Since 0% mortality was seen at 100 mg/L (nominal) in the complementary t est (with pH adjustment that yielded test water with an initial pH of 7.00), the 100% mortality at 100 mg/L (nominal) in the definitive test (with unadjusted pH) appears related to the acidification of the test solution (pH 3.28) by the ethanedioic acid. As indicated by the study authors, “...the observed effect [on fish mortality in the definitive test] can be considered to be related to the acid character of the test item.” [CiToxLAB 2013a, p. 20] Thus, the definitive test (testing without pH adjustment) provides the most conservative and appropriate evaluation of the toxicity of the test substance by reflecting the influence of its inherent acidity.

Validity Criteria : OECD TG 203 study validity criteria were met by fulfilling conditions for ≤ 10% mort ality in the control group, maintenance of environmental test conditions (except for decreases in pH and test substance concentration over the duration of the study) , dissolved oxygen concentration ≥ 60% of air saturation value throughout the tes t, and use of measured test
substance concentration s as the basis for results given that measured test concentrations were not maintained within ± 20% of nominal concentrations during the test.

CONCLUSIONS

For e thanedioic acid, hydrate (1:2), t he 96

- hour LC 50 for zebrafish based on mean measured concentrations in the definitive test (testing with unadjusted pH) was 56.0 mg/L. The equivalent

96

- hour LC 50 for e thanedioic acid (the designated test rule substance) was 39.8 mg/L.

STUDY RELIABILITY

[2] Relia ble with restrictions. The study w as limited by a failure to maintain test substance
concentrations throughout the test period. In the definitive test, the test solution was not stable
with losses of 41

- 76% from the initial measured concentrations for no minal concentrations of
6.25

- 50 mg/L (end

- of

- study concentrations were not measured at the 100 mg/L nominal concentration). In the stability tests conducted in ISO medium ( CiToxLAB 2013b ), the ratio of
final/initial measured concentrations after a 4

- day s torage period at room temperature was 112 and 115% for the 5 and 100 mg/L nominal concentrations, respectively, suggesting that the test substance would be stable under test conditions. The investigators did not provide a rationale for why the losses were so high in the fish study, as compared to the stability tests. In addition, the reliability of the LC 50 values could be affected by the lack of intermediate response levels, because the calculation is based on 100% response at the highest concentration a nd 0% response at lower levels and in controls (essentially a concentration

- response curve with two points).Final HPV 2 Chemical Data Adequacy Revie w CASRN 144

- 62

- 7

10 REFERENCE S

CiToxLAB Hungary Ltd. 2013a. Final Report. Acute toxicity test with ethanedioic acid, hydrate (1:2) [CAS RN 6153

- 56

- 6] (hydrated form of ethanedioic acid [CAS R N 144

- 62

- 7]) on Zebrafish ( Brachydanio rerio ). Study code 11/339

- 009H. Study Director: Krisztina Sipos. 19
March 2013.

CiToxLAB Hungary Ltd. 2013b. Final Report. Validation of the analytical method for the determination of ethanedioic acid, hydrate (1:2) [CAS RN 6153

- 56

- 6] (hydrated form of ethanedioic acid [CAS RN 144

- 62

- 7]). Study code 11/339

- 316AN. Study Director: Zsolt
Sarvari. 21 March 2013.

Text in quotes is taken directly from CiToxLAB (2013a).Final HPV 2 Chemical Data Adequacy Revie w CASRN 144

- 62

- 7

11 D

- 2

. Acute Toxicity to Aquatic Invertebra tes

Sum mary of endpoint: Adequate

Study 1: Acute toxicity study in Daphnia magna (CiToxLAB Hungary Ltd., 2013 c )

Title: Acute immobilization test with ethanedioic acid, hydrate (1:2) [CASRN 6153

- 56

- 6] (hydrated form of ethanedioic acid [CASRN 144

- 62

- 7 ]) on Daphnia ( Daphnia magna )

This study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Ethanedioic acid, hydrate (1:2), CASRN 6153

- 56

- 6

Remarks: Batch No. YP1

- 20120528C

. Ethanedioic acid (CASRN 144

- 62

- 7, aka oxalic acid) was tested in its hydrated (dihydrate) form, as white crystals that were 99.69% pure and supplied by the study sponsor. 1

Conversion factor for calculating mg of ethanedioic acid (C 2 H 2 O 4 ) from mg of the dihydrate (C 2 H 2 O 4 ·2H 2 O) = 0.71 [r atio of molecular weight of ethanedioic acid (90.03 g/mol) to ethanedioic acid dihydrate (126.07 g/mol)].

METHOD

Method/guideline followed: OECD TG 202 , “ Daphnia sp., Acute Immobilisation Test ” (2004);
OPPTS 850.1010

, “ Aquatic Invertebrate Acute Toxici ty Test, Freshwater Daphnids ” (1996)

Test type: Daphnia sp., acute immobilization test

- static conditions

GLP compliant?: Yes

Year study was performed: 201 2

Test species: Water flea ( Daphnia magna ) ; source: National Institute of Public Health, Budapest, Hungary.

Analytical monitoring: Yes

1 The certificate of analysis (COA) provided i n the study report identifies the test substance as oxalic acid (synonymous with ethanedioic acid), lot no. YP1

- 20120528C, but does not state whether the substance is in the anhydrous or dihydrate form. Furthermore, the composition provided in the COA doe s not list water as a component of the test substance, which suggests that the test substance was anhydrous. Therefore, although the test substance is identified as the dihydrate form of ethanedioic acid throughout the text of the study report, the COA
do es not confirm this identity of the test substance.Final HPV 2 Chemical Data Adequacy Revie w CASRN 144

- 62

- 7

12 Exposure duration: 48 hours

Concentrations a tested: Concentration range

- finding test (unadjusted pH) : Nominal: 0, 0.1, 1, 10

, and 100 mg/L Definitive test (Phase 1 , unadjusted pH ):
Nominal: 0, 6 .25, 12.5, 25, 50

, and 100 mg/L
Geometric mean measured b : 0, 4. 84 , 8.26, 11.64, 21.94

, and 73.65 mg/L Complementary test (Phase 2 , adjusted pH ):
Nominal: 0, 50 and 100 mg/L
Geometric mean measured b : 0, 58.26

, and 109.4 mg/L

a Concentrations are exp ressed as mg/L of e thanedioic acid, hydrate (1:2)

. b Geometric means of initial and 48

- hour concentrations.

Test method/conditions remarks: Preliminary Range

- finding T est (unadjusted pH) : Test substance concentrations in the definitive
test were based o n a preliminary test in which groups of 10 daphnids

, divided into two replicates of 5 animals each, were exposed to ethanedioic acid, hydrate (1:2) at nominal concentrations of 0, 0.1, 1, 10 or 100 mg/L under static conditions for 48 hours.

Definitive T est (Phase 1, unadjusted pH) : Twenty daphnids , divided into 4 replicates of five animals each, were exposed to nominal concentrations of 0, 6.25, 12.5, 25, 50

, or 100 mg/L under static conditions for 48 hours. Concentrations were measured at the beginnin g and end of testing at all exposure levels.

Complementary Test (Phase 2, adjusted pH) : Twenty daphnids , divided into 4 replicates of five animals each, were exposed to nominal concentrations of 0, 50

, or 100 mg/L under static
conditions for 48 hours. Concentrations were measured at the beginning and end of testing

.

The complementary test was conducted because pH values were below 6 in the test solution s at the two highest concentration s in the definitive (Phase 1) test. In particular, “Because

... the pH did not remain in the range 6

- 9 in the definitive test at the two highest concentration levels of 50 and 100 mg/L, a complementary study was carried out, adjusting the pH of the stock solution to
that of the dilution water before addition of the test s ubstance. The pH was adjusted through the addition of NaOH.” [CiToxLAB 2013c, p. 14] The complementary test therefore tested only 50 and 100 mg/L nominal concentrations at a pH within the range (6

- 9) recommended by OECD

TG 202. ( A fter adjustment, the p H of test solutions at nominal concentrations of 50 and 100 mg/L was ~7.7 at initiation of exposure)

.

Test solutions : “Reconstituted water (ISO medium, according to OECD 202) was used as dilution water for both the range finding and definitive tests [an d complementary study]. The same composition of reconstituted water was used for the tests and for breeding the test animals. Dilution water had a pH in the range of 6 to 9. ” [ CiToxLAB 2013c, p. 12] ISO medium had an approximate theoretical total hardn ess of 249 mg CaCO 3 /L.Final HPV 2 Chemical Data Adequacy Revie w CASRN 144

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13 A stock test solution of 100 mg/L was prepared prior to treatment by diluting 50 mg of test
substance in 500 mL of ISO medium. The test solutions were prepared by dilution of the stock
solution.

“In the complementary experiment t he pH of the stock solution was adjusted and then the appropriate dilution was prepared and distributed into test vessels prior to introduction of the Daphnia

. ” [CiToxLAB 2013c, p. 11]

Test conditions : S tatic conditions were used “since the test material was found to be stable in water over the test period

... ” [CiToxLAB 2013c, p. 8] during the analytical method validation. [see CiToxLAB 2013b, pp. 13

- 14] The validity of this statement is not supported by the analytical measurements conducted on samples fro m the definitive test . In particular, the problem with maintaining test concentrations throughout the study suggest s that a semi

- static or
flow

- through system should have been utilized.

The photoperiod was 16 hours light: 8 hours dark. During the test , animals were not fed, test vessels were not aerated, and test solution volume was at least 5 mL/animal (exact volume not specified).

Test organisms : Test Daphnia were less than 24 hours old at the beginning of tes ting and “ were derived from healthy s tock (showing no signs of stress such as high mortality, presence of males and ephippia, delay in the production of the first brood, discolored animals).” [CiToxLAB 2013c, p. 12] “There was no acclimatization because the water used was similar to the cul ture water.” [CiToxLAB 2013c, p. 12]

Observations : “ The immobility or mortality of the Daphnia was determined by visual observation
24 and 48 hours after the start of the test. Those animals not able to swim within 15 seconds after gentle agitation of the test beaker [were] considered to be immobile. The number of immobilised animals and the percentage of immobility were determined at 24 and 48 hours. The water temperature was measured at the start of the test and [at] 24

- hour intervals thereafter, ox ygen concentrations and pH of the controls and the test solutions were measured at the beginning and at the end of the experiments. [CiToxLAB 2013c, p. 14 ]

Analytical Monitoring : Concentrations of ethanedioic acid, hydrate (1:2) were analytically
determ ined using a validated HPLC

- UV method that is summarized in CiToxLAB 2013c (Appendix 4) and detailed in CiToxLAB 2013b. Samples (number not indicated) were taken for analysis at the beginning and end of the definitive and complementary tests.

Statistical evaluation : EC 50 values , based on measured concentrations, were calculated by Probit analysis using SPSS PC+ software

. The LOEC and NOEC values were determined directly from
the raw data.Final HPV 2 Chemical Data Adequacy Revie w CASRN 144

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14 R ESULTS

Endpoint value(s): All endpoint values are based on geometric mean measured concentrations (means of initial and 48

- hour concentrations).

Based on testing with unadjusted pH (definitive test):

Ethanedioic acid, hydrate (1:2 ), CASRN 6153

- 56

- 6 : 48

- hour EC 50 = 38.89 mg/L; 95% confidence interval = 28.0 5

- 61.70 mg/L
48

- hour LOEC = 73.65 mg/L
48

- hour NOEC = 21.94 mg/L

Ethanedioic acid , CASRN 144

- 62

- 7 ; c orresponding equivalent toxicity values were calculated
from the values for e thanedioic acid, hydrate (1:2) using a conversion factor of 0.71 (ratio of

m olecular weight of the acid to the dihydrate):

48

- hour EC 50 = 27. 61 mg/L; 95% confidence interval = 19.92

- 43.81 mg/L
48

- hour LOEC = 52.29 mg/L
48

- hour NOEC = 15.58 mg/L

Based on testing with adjusted pH (complementary test):

Ethanedioic acid, hydrate ( 1:2) , CASRN 6153

- 56

- 6 : 48

- hour EC 50 > 109.4 mg/L
48

- hour NOEC = 109.4 mg/L (highest concentration tested)

Ethanedioic acid , CASRN 144

- 62

- 7; equivalent toxicity value s : 48

- hour EC 50 > 77.7 mg/L
48

- hour NOEC = 77.7 mg/L (highest concentration tested )

Resu lts remarks:
Measured Test Concentrations : For the definitive test, nominal and mean measured
concentrations

, with percentage of nominal values

, are shown in the following table. Measured
concentrations declined 47

- 84% from the initiation of exposure t o the end of exposure.

M easured concentrations of ethanedioic acid, hydrate (1:2) with 95% confidence intervals
in the definitive test

Nominal
Concentration (mg/L) Measured Concentration at Start of Exposure Measured Concentration at End of Exposure

Concentration (mg/L) Percentage of Nominal (%) Concentration (mg/L) Percentage of Nominal (%)

0 Not detected

- Not detected

- 6.25 7.7 ± 0.09 123 3.1 ± 0.34 49

12.5 13.8 ± 0.66 110 5.0 ± 2.12 40

25 28.7 ± 2.2 115 4.7 ± 0.87 19

50 49.4 ± 2.14 99 9.7 ± 5.2 19Final HPV 2 Chemical Data Adequacy Revie w CASRN 144

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15 Nominal
Concentration (mg/L) Measured Concentration at Start of Exposure Measured Concentration at End of Exposure

Concentration (mg/L) Percentage of Nominal (%) Concentration (mg/L) Percentage of Nominal (%)

100 101 ± 1.26 101 53.7 ± 4.4 54

For nominal concentrations of 0, 6.25, 12.5, 25, 50

, and 100 mg/L, geometric mean measured
concentrations (means of initial and 48

- hour values) were 0, 4.84, 8.26, 11.64, 21.94

, and 73.65 mg/L, respectively. Th e rationale for determining geometric mean values was: “As the measured concentrations deviated more than 20 per cent from the nominal in most cases, biological results are based on the measured geometric mean concentrations.” [CiToxLAB 2013c, p. 14]. The geometric mean measured concentrations appear to have been calculated using start

- and end

- of

- study concentrations.

For the complementary test, geometric mean measured concentrations were 58.26 and 109.4 mg/L for nominal concentrations of 50 and 100 m g/L, respectively. Although concentrations were measured at the beginning and end of testing, measured concentrations at each of these time points were not reported (unlike the definitive test).

Results of the Preliminary R ange

- finding T est (unadjusted and unmeasured pH) : The incidence
of Daphnia immobilization was 100% at 100 mg/L and 0 % at ≤ 10 mg/L . Based on these data, five test concentrations ranging from 6.25 to 100 mg/L in a geometric series (separation factor of 2) and one untreated control wer e selected for the definitive test. Test substance concentrations and pH were not measured in the range

- finding test.

Results of the Definitive Test (with unadjusted pH ) : Incidences of Daphnia immobilization and pH values observed in the definitive test are shown in the following table.

Immob ilization incidence and pH value s observed in the definitive test

Nominal
Concentration (mg/L) Geometric Mean
Measured
Concentration (mg/L) Incidence of
Immobilization

at 48 Hours 1 Mean pH 2

0 Hours 48 Hours

0 0 0/20 7.61 7.46

6.25 4.84 0/20 7.66 7.21

12.5 8.26 0/20 7.53 7.14

25 11.64 0/20 7.25 7.07

50 21.94 0/20 5.47 5.97

100 73.65 20/20 3.21 3.27

1 Immobilization in four replicates combined

2 Mean of pH measurements from four replicates

The incidence of immobilization was 20/2 0 at 100 mg/L vs. 0/20 at all concentration s ≤ 50 mg/L, including the control. The pH of test water decreased with increasing test substance
concentration and was below pH 6 at the two highest nominal concentrations of 50 mg/L (pHFinal HPV 2 Chemical Data Adequacy Revie w CASRN 144

- 62

- 7

16 5.47

- 5.97) and 100 mg/L (pH 3.21

- 3.27)

. During the definitive test , water temperature ranged
from 20.4 to 21.1 ° C and dissolved oxygen concentration from 7.1 to 8.4 mg/L.

Results of the Complementary Test (with adjusted pH) : Incidences of Daphnia immobilization and pH values observed in the complementary test are shown in the following table.

Immob ilization incidence and pH value s observed in the complementary test

Nominal
Concentration (mg/L) Geometric Mean
Measured
Concentration (mg/L) Incidence of
Immobilization

at 48 Hours 1 Mean pH 2

0 Hours 48 Hours

0 0 0/20 7 .78 7.87

50 58.26 0/20 7.72 7.67

100 109.4 0/20 7.74 7.64

1 Immobilization in four replicates combined

2 Mean of pH measurements from four replicates

OECD TG 202 indicates that the acute test in Daphnia should initially be carried out without pH adjust ment but that a second test can be carried out if the pH does not remain in the range of 6 to 9. Because pH values fell below 6 for the two highest test substance concentrations of the definitive test, the complementary test was conducted by retesting the 50

- and 100

- mg/L
concentrations with the pH of stock solutions adjusted to the pH of the dilution water prior to exposure of the D aphnia to the test solutions

. Mean pH values of four replicates/test
concentration were 7.6

- 7.7 throughout the 48

- hour test period at both 50 and 100 mg/L compared
with 7.8

- 7.9 at 0 mg/L

. No immobilization of Daphnia was observed in this test. During the complementary test, water temperature ranged from 20.5 to 20.8 ° C and dissolved oxygen
concentration from 6.8 to 8.3 mg/L.

Selection of Data for Derivation of the EC 50 : S ince 0% immobility was seen at 100 mg/L (nominal) in the complementary test (with pH adjustmen t that yielded test water with a pH of

~ 7. 7), t he 100% immobility at 100 mg/L ( nominal ) in the definitive test (wi th unadjusted pH)

appears to be related to the acidi fication of the test solution (pH 3.2

- 3.3) by the ethanedioic acid. As indicated by the study authors, “the observed effect [on Daphnia immobility in the definitive test] can be considered to be related to the acid character of the test item.” [CiToxLAB 2013c , p. 18 ] Thus, the definitive test (testing without pH adjustment) provides the most conservative and appropriate evaluation of the toxicity of the test substance by reflecting the influence of its inherent acidity.

Validity criteria : OECD TG 202 study validity criteria were met by fulfilling conditions for ≤ 10% immobilization in the control group and dissolved oxygen concentration ≥ 3 mg/L at the
end of the test.Final HPV 2 Chemical Data Adequacy Revie w CASRN 144

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17 CONCLUSIONS

F or ethanedioic acid, hydrate (1:2) , t he 48

- hour EC 50 for Daphnia magna based on mean
measured concentrations in the definitive tes t (testing with unadjusted pH) was 38.89 mg/L

. The equivalent 48

- hour EC 50 for e thanedioic acid (designated test rule substance) was 27. 61 mg/L.

STUDY RELIABILITY

[2] Reliable with restrictions. T he study w as limited by a failure to maintain test subst ance concentrations throughout the test period. In the definitive test, the test solution was not stable
with losses of 47

- 84% from the initial measured concentrations. In the stability tests using the Daphnia test solution ( CiToxLAB 2013b ), losses after a 2

- day storage period at room
temperature were 11 and 0% at nominal concentrations of 5 and 100 mg/L , respectively, suggesting that the test substance would be stable under test conditions. The investigators did not provide a rationale for why the losse s were so high in the definitive test, as compared to the stability tests. In addition, the reliability of the EC 50 values could be affected by the lack of intermediate response levels, because the calculation is based on 100% response at the highest

conc entration and 0% response at lower levels and in controls (essentially a concentration

response curve with two points )

. Also, although the EC 50 value was reported with 95% confidence limits, the slope of the concentration

- response curve and its standard e rror were not provided as specified in OECD TG 202.

REFERENCE S

CiToxLAB Hungary Ltd. 2013b. Final Report. Validation of the analytical method for the determination of ethanedioic acid, hydrate (1:2) [CAS RN 6153

- 56

- 6] (hydrated form of ethanedioic acid [CAS RN 144

- 62

- 7]). Study code 11/339

- 316AN. Study Director: Zsolt
Sarvari. 21 March 2013.

CiToxLAB Hungary Ltd. 2013c. Final Report. Acute immobilisation test with ethanedioic acid, hydrate (1:2) [CAS RN 6153

- 56

- 6] (hydrated form of ethanedioic acid [CAS RN 144

- 62

- 7]) on Daphnia ( Daphnia magna ) . Study code 11/339

- 023DA. Study Director: Krisztina Sipos.
19 March 2013.

Text in quotes is taken directly from CiToxLAB (2013c).Final HPV 2 Chemical Data Adequacy Revie w CASRN 144

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18 D

- 3. Toxicity to Aquatic Plants

Summary of endpoint: Adequate

Study 1: Toxicity to Pseudokirchneri ella subcapitata ( CiToxLAB Hungary Ltd., 2013d )

Title: Growth inhibition test with ethanedioic acid, hydrate (1:2) [CASRN 6153

- 56

- 6] (hydrated form of ethanedioic acid [CASRN 144

- 62

- 7]) on algae ( Pseudokirchneriella subcapitata ).

This study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Ethanedioic acid, hydrate (1:2), CASRN 6153

- 56

- 6

Remarks: Batch No. YP1

- 20120528C. Ethanedioic acid (CASRN 144

- 62

- 7, aka oxalic acid) was tested in its hydrated (dihyd rate) form, as white crystals that were 99.69% pure and supplied by the study sponsor. 1

Conversion factor for calculating mg of ethanedioic acid (C 2 H 2 O 4 ) from mg of the dihydrate (C 2 H 2 O 4 ·2H 2 O) = 0.71 [ratio of molecular weight of ethanedioic acid (90.0 3 g/mol) to ethanedioic acid dihydrate (126.07 g/mol)].

METHOD

Method/guideline followed: OECD TG 201, “Freshwater Alga and Cyanobacteria, Growth Inhibition Test” (2011); Commission Regulation (EC) No 761/2009, C.3 “Freshwater Algae and Cyanobacteria, G rowth Inhibition Test” (2009); OPPTS 850.5400, “Algal Toxicity, Tiers I and II” (1996)

Test type: Growth inhibition in unicellular green algae

– static conditions

GLP compliant?: Yes

Year study was performed: 2012

Test species: Green algae, Pseudokirchneriella subcapitata (strain 61.81 SAG; identical to strains CCAP 278/4, UTEX 1648, and ATCC 22662); source: SAG, Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Germany.

1 The certificate of analysis (COA) provided in the study report identifies the test substance as oxalic acid (synonymous with ethanedioic acid), lot no. YP1

- 20120528C, but does not state whether the sub stance is in the anhydrous or dihydrate form. Furthermore, the composition provided in the COA does not list water as a component of the test substance, which suggests that the test substance was anhydrous. Therefore, although the test substance is ident ified as the dihydrate form of ethanedioic acid throughout the text of the study report, the COA does not confirm this identity of the test substance.Final HPV 2 Chemical Data Adequacy Revie w CASRN 144

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19 Analytical monitoring: Yes

Exposure dur ation: 72 hours

Concentrations a tested:

Range

- finding test (unadjusted pH): Nominal: 0, 0.1, 1, 10, and 100 mg/L Definitive test (Phase 1, unadjusted pH): Nominal: 0, 6.25, 12.5, 25, 50, and 100 mg/L
Geometric mean measured b : 0, 4.5, 7.9, 19.7, 44 .9, and 89.7 mg/L Complementary test (Phase 2, adjusted pH): Nominal: 0, 50, and 100 mg/L
Geometric mean measured b : 0, 34.2, and 97.2 mg/L

a Concentrations are expressed as mg/L of ethanedioic acid, hydrate (1:2).

b Geometric means of initial and 72

- hour concentrations.

Test method/conditions remarks:

Preliminary Range

- finding Test (unadjusted pH) : Test substance concentrations in the definitive test were based on a preliminary test in which algae were exposed to ethanedioic acid, hydrate (1:2) at nominal concentrations of 0, 0.1, 1, 10, or 100 mg/L under static conditions for 72 hours. Two replicates per exposure concentration and 3 replicates for controls were tested.

Definitive Test (Phase 1, unadjusted pH) : Exponentially growing algal c ultures were exposed to nominal concentrations of 0, 6.25, 12.5, 25, 50, or 100 mg/L under static conditions for 72 hours. Testing was conducted using 3 replicates per exposure concentration and 6 replicates for
controls. Concentrations were measured at t he beginning and end of testing.

Complementary Test (Phase 2, adjusted pH): Exponentially growing algal cultures were exposed to nominal concentrations of 0, 50, or 100 mg/L under static conditions for 72 hours. Testing was conducted using 6 replic ates for each exposure concentration and 6 replicates for controls. Concentrations were measured at the beginning and end of testing.

The complementary test was conducted because initial pH values of test solutions at the two highest concentrations in th e definitive test were more than 3 units below that of the control value. The study authors stated “Because there was evidence of marked change in the pH of the test water after addition of the test substance, and the pH was below 6 in the definitive test at the two highest concentrations of 50 and 100 mg/L, a complementary study was carried out, adjusting the pH of the stock solution to that of the dilution water before addition of the test substance. The pH was adjusted through the addition of NaOH.” [ CiToxLAB 2013d, p. 15] The complementary test therefore tested only 50 and 100 mg/L nominal concentrations in test solutions that had initial pH values similar to that of the control test solution. (With adjustment, the pH of test solutions at initiation of exposure at nominal concentrations of 50 and 100 mg/L

was 7.70

- 7.80; pH of the control solution was 7.82).Final HPV 2 Chemical Data Adequacy Revie w CASRN 144

- 62

- 7

20 Test Solutions : “Reconstituted algal growth medium (OECD medium, according to OECD 201) was used as dilution water for the range finding, defin itive test and also for the complementary study.” [ CiToxLAB 2013d, p. 12] A pH value of 8.1 is specified for OECD growth medium by

OECD TG 201.

In the definitive test (Phase 1), “An amount of 100 mg test item was dissolved in one litre vehicle (OECD med ium) by mechanical dispersion in order to give the 100 mg/L test concentration. The test solutions were prepared by the appropriate diluting of this stock solution and distributed into test vessels prior to introduction of algae.” [CiToxLAB 2013d, p. 11]

“In the complementary experiment (Phase 2) the pH of the stock solution was adjusted and then the appropriate dilution was prepared and distributed into test vessels prior to introduction of algae.” [CiToxLAB 2013d, p. 11]

Test Conditions : In the defi nitive and complementary tests, “The algal culture flasks were continuously illuminated. The light intensity at the position occupied by algal culture flasks
duri ng the test was about 8116 lux (equivalent to 110 μE/m 2 /s), which was ensured with fluorescent lamps (with a spectral range of 400

- 700 nm).” [CiToxLAB 2013d, p. 13]

Test Organisms : “The pre

- culture was intended to give an amount of alga suspension suita ble for the inoculation of test cultures. The pre

- culture was incubated under the conditions of the study in an aerated Algal Growth Medium and used when still exponentially growing (after an incubation period of 3 days). The cell count of [the] above cu lture was determined by [a] microscopic method and this cell suspension was diluted with Algal Growth Medium to 10 7
cells/mL.” [CiToxLAB 2013d, p. 13]

Test Procedure : “The exposure time was 72 hours. The test was started (Day 0) by inoculation
of a bio mass of approximately 10 4 algal cells per mL test medium in both... the definitive and complementary studies... Volumes of 100 mL algal suspension per replicate in 250 mL Erlenmeyer flasks were continuously shaken by a laboratory orbital shaker to keep algae in suspension. The flasks were covered with air

- permeable stoppers.” [CiToxLAB 2013d, p. 14]

Observations : “The cell numbers were determined at 24, 48 and 72 hours after the treatment by manual cell counting using a microscopic method with a counting c hamber. Microscopic observation of the algal cells [at] each concentration and in the control was performed (at 24h, 48h and 72h) to detect any abnormal appearance of the algae.” [CiToxLAB 2013d, p. 15]

For the definitive and complementary tests, pH was measured in all exposed and control groups at the beginning and end of testing. “Culture temperature was checked at the beginning of the study and each day thereafter in a flask filled with water, in the climatic chamber. In addition,
temperature was co ntinuously measured (with a min/max thermometer) within the climate chamber.” [CiToxLAB 2013d, p. 13]

Analytical Monitoring : Concentrations of ethanedioic acid, hydrate (1:2) were analytically determined at the beginning and end of testing using a valid ated HPLC

- UV method asFinal HPV 2 Chemical Data Adequacy Revie w CASRN 144

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- 7

21 summarized in CiToxLAB 2013d (Appendix 5) and detailed in CiToxLAB 2013b. Samples (number not indicated) were taken for analysis at the beginning and end of the definitive and complementary tests.

Calculations and Statistical Analy sis : Growth inhibition was evaluated by statistical comparisons of 0

- 72 hour average specific growth rates, yield, and biomass (area under growth curves) in treated and control groups.

“The section

- by

- section specific growth rates in the control cultures were assessed (calculated as the specific growth rates for each day during the course of the test (days 0

- 1, 1

- 2 and 2

- 3)) to demonstrate exponential growth for the entire study period.” [CiToxLAB 2013d, p. 18]

“The E
r C 50 , E b C 50 and E y C 50 values were de termined using Probit analysis by SPSS PC+
software.” [CiToxLAB 2013d, p. 18]

“Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using analysis of variance (ANOVA) and Bonferr oni t

- Test and Dunnett’s Test (α = 0.05) by TOXSTAT software.” [CiToxLAB 2013d, p. 18] These comparisons were used to determine the LOEC and NOEC.

RESULTS

Endpoint value(s): All endpoint values are based on geometric mean measured concentrations (mean s of initial and 72

- hour concentrations)

Based on testing with unadjusted pH (definitive test):

Ethanedioic acid, hydrate (1:2), CASRN 6153

- 56

- 6 : 72

- h EC 50 (growth rate) = 27.17 mg/L; 95% confidence interval = 25.21

- 29.70 mg/L

72

- h LOEC (growth rate ) = 19.7 mg/L

72

- h NOEC (growth rate) = 7.9 mg/L

72

- h EC 50 (yield) = 24.28 mg/L; 95% confidence interval = 22.70

- 26.49 mg/L

72

- h LOEC (yield) = 19.7 mg/L

72

- h NOEC (yield) = 7.9 mg/L

72

- h EC 50 (biomass) = 24.28 mg/L; 95% confidence interval = 22.70

- 26.49 mg/L

72

- h LOEC (biomass) = 19.7 mg/L

72

- h NOEC (biomass) = 7.9 mg/L

Ethanedioic acid, CASRN 144

- 62

- 7; c orresponding equivalent toxicity values were calculated
from the values for e thanedioic acid, hydrate (1:2) using a conversion factor of 0.71 (ratio of molecular weight of the acid to the dihydrate):

72

- h EC 50 (growth rate) = 19.29 mg/L; 95% confidence interval = 17.90

- 21.09 mg/L

72

- h LOEC (growth rate) = 14.0 mg/L

72

- h NOEC (growth rate) = 5.6 mg/LFinal HPV 2 Chemical Data Adequacy Revie w CASRN 144

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- 7

22 72

- h EC 50 (yield) = 17.24 mg/L; 95% confidence interva l = 16.12

- 18.81 mg/L

72

- h LOEC (yield) = 14.0 mg/L

72

- h NOEC (yield) = 5.6 mg/L

72

- h EC 50 (biomass) = 17.24 mg/L; 95% confidence interval = 16.12

- 18.81 mg/L

72

- h LOEC (biomass) = 14.0 mg/L

72

- h NOEC (biomass) = 5.6 mg/L

Based on testing with adjusted pH (complementary test):

Ethanedioic acid, hydrate (1:2), CASRN 6153

- 56

- 6 : 72

- h EC 50 (growth rate) > 34.2 mg/L

72

- h LOEC (growth rate) = 34.2 mg/L

72

- h NOEC (growth rate) < 34.2 mg/L

72

- h EC 50 (yield) > 34.2 mg/L

72

- h LOEC (yield) = 34.2 mg/L

72

- h NOEC (yield) < 34.2 mg/L

72

- h EC 50 (biomass) > 34.2 mg/L

72

- h LOEC (biomass) = 34.2 mg/L

72

- h NOEC (biomass) < 34.2 mg/L

Ethanedioic acid, CASRN 144

- 62

- 7; equivalent toxicity values:

72

- h EC 50 (growth rate) > 24.3 mg/L

72

- h LOEC (growth rate) = 24.3 mg/ L
72

- h NOEC (growth rate) < 24.3 mg/L

72

- h EC 50 (yield) > 24.3 mg/L

72

- h LOEC (yield) = 24.3 mg/L

72

- h NOEC (yield) < 24.3 mg/L

72

- h EC 50 (biomass) > 24.3 mg/L

72

- h LOEC (biomass) = 24.3 mg/L

72

- h NOEC (biomass) < 24.3 mg/L

Results remarks:

Measured Te st Concentrations : For the definitive test, nominal and mean measured concentrations, with percentage of nominal values, are shown in the following table. Measured
concentrations declined 13

- 66% from the initiation of exposure to the end of exposure.Final HPV 2 Chemical Data Adequacy Revie w CASRN 144

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23 Me asured concentrations of ethanedioic acid, hydrate (1:2) with 95% confidence intervals
in the definitive test

Nominal
Concentration (mg/L) Measured Concentration at Start of Exposure Measured Concentration at End of Exposure

Concentration (mg/L) Perce ntage of Nominal (%) Concentration (mg/L) Percentage of Nominal (%)

0 Not detected

- Not detected

- 6.25 7.5 ± 0.32 120 2.7 ± 0.36 43

12.5 13.6 ± 0.49 109 4.6 ± 3.85 37

25 25.1 ± 0.79 100 15.4 ± 2.24 62

50 49.2 ± 0.86 98 40.9 ± 1.12 82

100 96.4 ± 2.7 7 96 83.4 ± 11.3 83

For nominal concentrations of 0, 6.25, 12.5, 25, 50, and 100 mg/L, corresponding geometric mean measured concentrations (means of initial and 72

- hour values) in the definitive study were 0, 4.5, 7.9, 19.7, 44.9, and 89.7 mg/L, respect ively. The rationale for providing these values is as follows: “As the analytically measured concentration deviated more than 20 per cent from the nominal in most cases, biological results are based on the measured geometric mean concentrations.” [CiTox LAB 2013d, p. 15]. The geometric mean concentrations appear to have been calculated using start

- and end

- of

- study concentrations.

For the complementary test, geometric mean measured concentrations were 34.2 and 97.2 mg/L
for nominal concentrations of 50 and 100 mg/L, respectively. Although concentrations were measured at the beginning and end of testing, measured concentrations at each of these time points were not reported (unlike the definitive test).

Results of the Preliminary Range

- finding Test (una djusted and unmeasured pH) : Inhibition of algae biomass (based on number of cells/mL after 72 hours) was ~99% at 100 mg/L and ~3

- 4%

at ≤ 10 mg/L. Based on these data, five nominal test concentrations in a geometric series (separation factor of 2.0) ranging from 6.25 to 100 mg/L were selected for the definitive test. Test substance concentrations and pH were not measured in the range

- fin ding test.

Results of the Definitive Test (with unadjusted pH) : Algae growth inhibition and pH values observed in the definitive test are shown in the following table.

Algae Growth Inhibition and pH values observed in the definitive test

Nominal

Conc entration (mg/L) Geometric
Mean Measured

Concentration (mg/L) Mean % Inhibition 1
(0

- 72 hours) Mean pH 2

Growth

Rate Biomass Yield 0 h 72 h

0 0

- - - 7.87 8.36

6.25 4.5

- 0.4

- 1.9

- 1.9 7.72 8.64

12.5 7.9

- 0.5

- 1.2

- 1.9 7.54 8.59

25 19.7 4.7* 18* 18* 7. 00 8.47Final HPV 2 Chemical Data Adequacy Revie w CASRN 144

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24 Nominal

Conc entration (mg/L) Geometric
Mean Measured

Concentration (mg/L) Mean % Inhibition 1
(0

- 72 hours) Mean pH 2

Growth

Rate Biomass Yield 0 h 72 h

50 44.9 100* 100* 100* 4.64 4.85

100 89.7 100* 100* 100* 3.57 3.68

1 Percent inhibition, relative to control values (using mean of all replicates)

2 Mean of pH measurements from all replicates

* Statistically significantly different from the con trol (p < 0.05)

Inhibition of response parameters (growth rate, biomass, and yield) was 100% at 50 and 100 mg/L, 5

- 18% at 25 mg/L, and 0 % at ≤ 12.5 mg/L. “Thin cells were observed at the two highest examined concentrations of 50 and 100.0 mg/L (nominal), 48 and 72 hours after the start of the test in the definitive test (Phase 1).” [CiToxLAB 2013d, p. 23] The pH of test water decreased with increasing test substance concentration and was below pH 5 at the two highest nominal concentrations of 50 mg/L (pH 4.64

- 4.85) and 100 mg/L (pH 3.57

- 3.68). During the definitive test, daily measurements of temperature in a water

- filled flask within t he climate chamber ranged from 22.1 to 22.3°C; continuous measurements of temperature within the climate chamber ranged from 21.8 to 22.9°C.

Results of the Complementary Test (with adjusted pH) : Algae growth inhibition and pH values observed in the comp lementary test are shown in the following table.

Algae Growth Inhibition and pH values observed in the complementary test

Nominal
Concentration (mg/L) Geometric
Mean Measured

Concentration (mg/L) Mean % Inhibition 1
(0

- 72 hours) Mean pH 2

Growth

Rate Bi omass Yield 0 h 72 h

0 0

- - - 7.82 9.14

50 34.2 2.2* 8.0* 9.3* 7.80 9.06

100 97.2 5.5* 27.8* 13.0* 7.70 9.02

1 Percent inhibition, relative to control values (using mean of all replicates)

2 Mean of pH measurements from all replicates

* Statistically significantly different from the control (p < 0.05)

Because initial pH values of test solutions in the definitive test at the two highest concentrations of 50 mg/L (pH 4.64) and 100 mg/L (pH 3.57) were more than 3 units below that in the control (pH 7.87) , the complementary test was conducted by retesting the 50

- and 100

- mg/L concentrations with adjustment of the pH of stock solutions to that of the OECD TG 201 growth medium. Mean pH values at 0 hours in the complementary test were 7.82, 7.80, and 7.70 at nominal concentrations of 0, 50, and 100 mg/L, respectively. Inhibition of response parameters (growth rate, biomass, and yield) was 5.5

- 27.8% at 100 mg/L and 2.2

- 9.3% at 50 mg/L. During the complementary test, daily measurements of temperature in a wat er

- filled flask within the climate chamber ranged from 22.5 to 22.8 °C; continuous measurements of temperature within the climate chamber ranged from 22.1 to 23.3°C

.Final HPV 2 Chemical Data Adequacy Revie w CASRN 144

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25 Selection of Data for Derivation of the EC 50 Values : Since inhibition of algae growth p arameters in the complementary test (with pH adjustment that yielded test media with an initial pH of 7.7

- 7.8) at 50 and 100 mg/L was 2.2

- 5.5% for growth rate, 8.0

- 27.8% for biomass, and 9.3

- 13.0% for yield, the 100% inhibition of all three parameters at 5 0 and 100 mg/L in the definitive test appears to be primarily related to acidification of the test media (initial pH values were 4.64 and 3.57 at 50 and 100 mg/L, respectively) by the ethanedioic acid. However, the partial inhibition of these algae growth parameters that occurred under conditions in which test media had initial pH ≥ 7 (as seen at 50 and 100 mg/L in the complementary test and at 25 mg/L in the definitive test) suggests that ethaned i oic acid also exerts toxicity to algae that is related to m olecular features other than acidity. Thus, the definitive test (testing without pH adjustment) provides the most conservative and appropriate evaluation of the toxicity of ethanedioic acid by reflecting the influence of both its acidity and other inheren t molecular characteristics on inhibition of algae growth.

Validity Criteria : OECD TG 201 validity criteria were met by fulfilling conditions for increase of algae biomass in control cultures by a factor ≥ 16 within 72 hours (actual increase of 63.17x in definitive test and 73.00x in complementary test), a mean coefficient of variation of ≤ 35% for

section

- by

- section (days 0

- 1, 1

- 2, and 2

- 3) specific growth rates in controls (actual value of
21.47% in defi nitive test and 17.16% in complementary test), and a coefficient of variation of average specific growth rates of ≤ 7% during the whole test period in replicate control cultures (actual value of 0.82% in definitive test and 0.88% in complementary test).

CONCLUSIONS

E thanedioic acid, hydrate (1:2): The 72

- hr EC 50 was 27.17 mg/L for growth rate and 24.28 mg/L for yield and biomass based on mean measured concentrations in the definitive test (testing with unadjusted pH). The 72

- hour LOEC for growth rate, yield, and biomass was 19.7 mg/L. The

72

- hour NOEC for growth rate, yield, and biomass was 7.9 mg/L.

E thanedioic acid (designated test rule substance): The equivalent 72

- hr EC 50 was 19.29 mg/L for growth rate and 17.24 mg/L for yield and biomass. The 7 2

- hour LOEC for growth rate, yield, and biomass was 14.0 mg/L. The 72

- hour NOEC for growth rate, yield, and biomass was 5.6 mg/L.

STUDY RELIABILITY

[2] Reliable with restrictions. The study was limited by a failure to maintain test substance

concentrat ions throughout the test period. In the definitive test, the test solution was not stable
with losses of 13

- 66% from the initial measured concentrations. In the stability tests using the algae test solution (CiToxLAB 2013b), losses after a 3

- day storage period at room temperature were 6 and 0% at nominal concentrations of 5 and 100 mg/L, respectively, suggesting that the test substance would be stable under test conditions. The investigators did not provide a rationale for why the losses were so high in the definitive test in algae, as compared to the stability tests.Final HPV 2 Chemical Data Adequacy Revie w CASRN 144

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26 REFERENCE S

CiToxLAB Hungary Ltd. 2013b. Final Report. Validation of the analytical method for the determination of ethanedioic acid, hydrate (1:2) [CAS RN 6153

- 56

- 6] (hydrated form of ethanedioic acid [CAS R N 144

- 62

- 7]). Study code 11/339

- 316AN. Study Director: Zsolt
Sarvari. 21 March 2013.

CiToxLAB Hungary Ltd. 2013d. Final Report. Growth inhibition test with ethanedioic acid, hydrate (1:2) [CAS RN 6153

- 56

- 6] (hydrated form of ethanedioic acid [CAS RN 144

- 62

- 7]) on algae ( Pseudokirchneriella subcapitata ). Study code: 11/339

- 022AL. Study Director: Krisztina Sipos. 19 March 2013.

Text in quotes is taken directly from CiToxLAB (2013d).Final HPV 2 Chemical Data Adequacy Revie w CASRN 144

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27 D

- 4 . Genetic Toxicity

– Chromosomal Aberrations

Summary of endpo int: Adequate

Study 1: Mammalian Chromosome Aberration Test in vitro (CiToxLAB Hungary Ltd., 2013e)

Title: In vitro mammalian chromosome aberration test of ethanedioic acid, hydrate (1:2) [CASRN 6153

- 56

- 6] (hydrated form of ethanedioic acid [CASRN 14 4

- 62

- 7])

Th is study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Ethanedioic acid, hydrate (1:2), CASRN 6153

- 56

- 6

Remarks: Batch No. YP1

- 20120528C. E thanedioic acid (CASRN 144

- 62

- 7, aka oxalic acid)

wa s tested in its hydrated (dihydrate) form, as white crystals that were 99.69% pure and supplied by the study sponsor. 1

Conversion factor for calculating μ g of ethanedioic acid (C 2 H 2 O 4 ) from μ g of the dihydrate (C 2 H 2 O 4 ·2H 2 O) = 0.71 [ratio of molecular we ight of ethanedioic acid (90.03 g/mol) to ethanedioic acid dihydrate (126.07 g/mol)].

METHOD

Method/guideline followed: OECD TG 473, “ In Vitro Mammalian Chromosome Aberration

Test ” ( 1997 ); Commission Regulation (EC) No 440/2008, B.10

, “ Mutagenicity

– In Vitro
Mammalian Chromosome Aberration Test ” (2008); OPPTS 870.5375, “ In Vitro Mammalian
Chromosome Aberration Test ” ( 1998 )

Test type: In vitro mammalian chromosomal aberration test

GLP compliant?: Yes

Year study performed: 2012

- 2013

Test species: Chinese hamster V79 lung cells ; supplier: ECACC (European Collection of Cell Cultures) ; ECACC Cat. No. 86041102 ; Lot No. 10H016 ; m orphology: f ibroblast

Metabolic activation: With and without

1 The certificate of analysis (COA) provided in the study report identifies the test substance as oxalic acid ( synonymous with ethanedioic acid), lot no. YP1

- 20120528C, but does not state whether the substance is in the anhydrous or dihydrate form. Furthermore, the composition provided in the COA does not list water as a component of the test substance , whic h suggests that the test substance was anhydrous . Therefore, although the test substance is identified as the dihydrate form of ethanedioic acid throughout the text of the study report, the COA
does not confirm this identity of the test substance.Final HPV 2 Chemical Data Adequacy Revie w CASRN 144

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28 Concentrations tested: Preliminary concentration selecti on assays: 0 (untreated control); 0 (vehicle controls; 1, 2.5 and 5% phosphate buffered saline); 0.064 , 0.32, 1.6, 8, 40, 200, 1000, 2500, and 5000 μg/mL (with and without S9).

Main chromosome aberration assays: 0 (vehicle control ; 1% phosphate buffere d saline); 12.5, 25, 50, 75, 100, 125, 150, 200

, and 400 μg/mL (with and without S9).

Statistical methods used: Fisher’s exact test. “The parameter evaluated for statistical analysis
was the number of cells with one or more chromosomal aberrations excl uding gaps.” [CiToxLAB 2013e, p. 23]

Test method/conditions remarks:
Formulations: “Due to the acidic property of the test item, formulations were prepared in a sufficient strength supplementary buffering system (phosphate buffered saline, PBS) to ens ure a suitable final pH at testing; the same vehicle was used in the negative controls.” 1 [CiToxLAB 2013e, p. 13]

“A supplementary pH buffering in the system was considered not to adversely affect the ability of the test item to cause genotoxic effects in the test system. The pH of the dosing solution was measured before treatment to prove that the amount of buffering did not result in unphysiological conditions (ie. alkalinity).” [CiToxLAB 2013e, p. 14]

Dosing solutions were prepared in PBS by dilut ing a 100 mg/mL stock formulation of the test substance immediately before treatment of cells.

The study report stated that: “Purity conversion was applied in the main experiments (ie. concentration of the test item in the dosing formulation was adju sted according to the water content of the provided material) as agreed by the Sponsor. For the purity conversion, the 74.0% purity data of the active ingredient (molecular weight of the dihydrate form: 121.6; molecular weight of the anhydrous form: 90.0 ) was taken into consideration.” 2 [CiToxLAB 2013e, p. 14] However, it is unclear whether reported test concentrations were given in units of the anhydrous or dihydrate form because the experimental design on p. 19 of the study report
indicates that test concentrations of 125 to 400 μg/mL refer to the dihydrate form of ethanedioic acid, suggesting that the purity conversion has not been applied.

Positive controls: Tests without S9 activation used 0.4 μL/mL (20

- hour treatment) or 1.0 μL/mL (3

- hour treatme nt) ethyl methanesulfonate (EMS) in Dulbecco’s Modified Eagle’s Medium

1 The s tudy report appears to indicate that a 1% PBS solution was used as the vehicle for test substance concentrations up to 1000 μg/mL, a 2.5% PBS solution was used as the vehicle for the test substance concentration of 2500 μg/mL, and a 5% PBS solution was used as the vehicle for the test substance concentration of 5000 μg/mL. Corresponding vehicle controls of 1, 2.5, and 5% PBS were used.

2 The molecular weight is incorrectly reported as 121.6 on p. 14 of the study report. The correct molecular weight of the dihydrate form of ethanedioic acid is 126.1, resulting in a conversion factor of 0.71 or 71%.Final HPV 2 Chemical Data Adequacy Revie w CASRN 144

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29 ( DMEM ) for the positive controls. Tests with S9 activation used 6.0 μg/mL cyclophosphamide monohydrate in sterile physiological saline solution (0.9% NaCl) for the positive controls.

Metabolic activation: A “ cofactor

- supplemented post

- mitochondrial S9 fraction prepared from
activated rat liver was used as an appropriate external metabolic activation system. ” [CiToxLAB 2013e, p. 16] Phenobarbital and β

- naphthoflavone were used as hepatic enzyme inducers.

Preliminary test: Results of a preliminary c ytotoxicity test were used to determine treatment concentrations for the main study. The preliminary test was conducted as two separate assays in
which c oncentrations of 0.064 , 0.32, 1.6, 8, 40, 200, 1000, 2500, and 5000 μg/mL were tested. Cells were treated for 3 hours with or without S9 (Assay A) or for 20 hours without S9 (Assay B), and harvested 20 hours after beginning of treatment in both assays. S ingle cultures were
tested for each test concentration and for negative (untreated and vehicle ) controls ; no positive
controls were included in either preliminary assay. The pH and osmolality of test medium were measured at the end of the treatment period

. The number of surviving cells was determined using a haemocytometer at the scheduled harvesting time.

Main study: The chromosome aberration assays were conducted as two independent experiments (Assays 1 and 2) with test concentrations of 12.5, 25, 50, 75, 100, 125, 150, 200

, and 400 μg/mL. Cells were treated for 3 hours with or without S9 (Assay 1), or for 3 hours with S9 or 20 hours without S9 (Assay 2), and harvested 20 hours after the beginning of the treatment in both cases. Duplicate cultures were tested for each test concentra tion and for negative (vehicle) and positive controls, although no untreated controls were included in either assay. Cytotoxicity was measured in a concurrent (third) culture.

Solubility (precipitation) of test substance in final test medium was visually examined at the beginning and end of treatment, and pH and osmolality of test medium were measured at the end
of treatment

.

The negative (vehicle) control, positive control

, and at least 3 test substance concentration s (based on observed solubility and/o r cytotoxicity results) from each assay were selected for chromosomal aberration analysis. A t least 200 well

- spread metaphase cells (from both duplicate cultures combined) were analyzed (or until a clear positive response was detected) for each
selected t est concentration and negative and positive control s

.

“At least one hundred metaphases with 22 ± 2 chromosomes (dicentric chromosomes were counted as two chromosomes) from each culture were examined for the presence or absence of
chromosomal aberrations (approximately 1000x magnification), where possible. Chromatid and
chromosome type aberrations (gaps, deletions and exchanges) were recorded separately.” [CiToxLAB 2013e, p. 21] Additionally, the number of polyploid and endoreduplicated cells was

recor d ed

.

The test substance was considered to be positive for induction of chromosomal aberrations if all of the following criteria were met:

• The frequency of metaphases with aberrant chromosomes (excluding gaps) was increased
at one or more test concentrati ons.Final HPV 2 Chemical Data Adequacy Revie w CASRN 144

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30 • The increases we re reproducible between replicate cultures and between tests with
identical treatment conditions.

• The increases we re statistically significant.

• The increases we re not associated with large changes in pH or osmolality in the treated
cul tures. (Large changes are defined as a difference of > 0.5 in pH units, compared to the vehicle control, or a difference of > 50 mmol/kg in osmolality, compared to the vehicle control.)

RESULTS

Genotoxicity result: Negative with or without metaboli c activation

Cytotoxic concentration (if applicable):
3

- hour treatment without activation: ≥ 100 μg/mL

20

- hour treatment without activation: ≥ 75 μg/mL

3

- hour treatment with activation: ≥ 100 μg/mL

Results remarks: Preliminary Test: Under all tes t conditions (3

- or 20

- hour treatment without S9 or 3

- hour treatment with S9), cell survival was ≥ 60% at test concentrations ≤ 40 μg/mL ; cell survival was < 10% at test concentrations ≥ 200 μg/mL

.

Chromosome Aberration Assay 1: This assay used a 3

- hour treatment with and without metabolic activation. No compound insolubility or large changes in pH or osmolality were
detected. The pH of test medium was 7.2

- 7.4 at the end of treatment. Cytotoxicity (less than 50% cell survival, relative to controls) was observed at 400, 200, 150, 125

, and 100 μg/mL in the test without S9 (relative cell survival of 7, 10, 18, 28

, and 30%, respectively), and at 400, 200,
150

, and 125 μg/mL in the test with S9 (relative cell survival of 7, 6, 24

, and 46%, respectively). Based on these findings, the conce ntrations selected for chromosomal aberration evaluation were 100, 75, 50

, and 25 μg/mL tested without S9 and 125, 100

, and 50 μg/mL tested with S9.

None of the evaluated test concentrations, with or without metabolic activation, caused a
statisticall y significant increase in the number of cells with structural chromosomal aberrations (excluding gaps) compared to the negative control group. Although Fisher’s exact test was used for statistical analysis, the resulting p

- value was not specified.

Ch romosome Aberration Assay 2: This assay used a 3

- hour treatment with metabolic activation
and a 20

- hour treatment without metabolic activation. Consistent with Assay 1, no compound insolubility or large changes in pH or osmolality were detected. The pH of test medium was 7.4
at the end of treatment. Cytotoxicity was observed at 400, 200, 150, 125, 100

, and 75 μg/mL in the test without S9 (relative cell survival of 4, 2, 7, 14, 12

, and 24%, respectively), and at 400, 200, 150, 125

, and 100 μg/mL in the t est with S9 (relative cell survival of 6, 11, 14, 30

, and 47%, respectively). Based on these cytotoxicity findings, the concentrations selected for chromosomal
aberration evaluation were 100, 75, 50

, and 25 μg/mL tested without S9 and 100, 75

, and 50 μg/m L tested with S9.Final HPV 2 Chemical Data Adequacy Revie w CASRN 144

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31 None of the evaluated test concentrations, with or without metabolic activation, caused a statistically significant increase in the number of cells with structural chromosomal aberrations (excluding gaps) compared to the negative cont rol group. Although Fisher’s exact test was used for statistical analysis, the resulting p

- value was not specified

.

Polyploid and /or endoreduplicated metaphases were observed at low frequencies in some negative control, treated

, and positive control sam ples in both assays , but their presence had no bearing on the conclusions of this study

.

Control data: Spontaneous aberration frequencies in the negative (vehicle) control groups of both assays were acceptable in comparison with the historical control range of the testing laboratory. “ In Assay 2, the mean aberration frequency in the negative control was 6.0% (experiment without metabolic activation) and 5.5% (experiment with metabolic activation), thus slightly above the upper limit of the general hist orical control range (0

- 5%) . However , this
difference was minor and considered to be acceptable. ” [CiToxLAB 2013e, p. 27]

The positive controls in both studies showed the expected increases in number of cells with
structural chromosome aberrations

. Res ults from positive controls were within the range of historical control data provided in the study report.

Validity of study : This study fulfilled the following conditions for a valid study: acceptable

responses in negative and positive control group s a nd selection of at least three test
concentrations for evaluation of chromosomal aberrations with an appropriate range of cytotoxicity (from ≤ 50% cell survival to little or no cytotoxicity).

CONCLUSIONS

Ethanedioic acid, hydrate (1:2) did not induce a statistically significant increased frequency of

ce lls with structural chromosomal aberrations (excluding gaps) when tested with or without S9 metabolic activation at concentrations below cytotoxic levels.

STUDY RELIABILITY

[ 2 ] Reliable with restrictions

. It is unclear whether test concentrations prese nted in the study report were given in units of the anhydrous or dihydrate form of ethanedioic acid. Also, the study authors provided an incorrect conversion factor of 0.74 (rather than 0.71) for calculating the concentration of anhydrous ethanedioic acid from the dihydrate form; if this conversion factor was used to calculate the reported concentrations, then the values are not accurate. Regardless of the units of the test concentrations, this study provides sufficient information to
reach a conclusion r egarding the ability of ethanedioic acid to induce chromosomal aberrations
in mammalian cells in vitro

.Final HPV 2 Chemical Data Adequacy Revie w CASRN 144

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32 REFERENCES

CiToxLAB Hungary Ltd. 2013e. In vitro Mammalian Chromosome Aberration Test of Ethanedioic acid, hydrate (1:2) [CAS RN 6153

- 56

- 6] (hyd rated form of ethanedioic acid [CAS

RN 144

- 62

- 7]). Study code: 11/339

- 020C. Study Director: Judit Hargitai. 22 March 2013.

Text in quotes is taken directly from CiToxLAB (2013e).