SRC TR-13-052

HPV2 TEST RULE DATA ADEQUACY REVIEW (FINAL): 

ACUTE TOXICITY TO FISH, ACUTE TOXICITY TO DAPHNIA, TOXICITY TO ALGAE,

AND CHROMOSOMAL ABERRATIONS

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, 2013
Final HPV2 Chemical Data Adequacy Review CASRN 144-62-7

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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 – Chromosomal Aberrations................................................................ 27
Final HPV2 Chemical Data Adequacy Review CASRN 144-62-7

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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 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 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 testing if log 

Kow < 4.2 or chronic toxicity to Daphnia and toxicity to algae testing if log Kow ≥ 4.2) and E2 

(chromosomal aberration or micronucleus test). Given that the log Kow of CASRN 144-62-7 has 

been reported to be less than zero1, the C1 aquatic toxicity 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 following 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 cells

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), the 

submitted tests were conducted using the 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 hygroscopic, 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 the 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 toxicity 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), Handbook of Environmental Data on Organic 

Chemicals, 4th 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).
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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 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. 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 behalf 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 144-62-7 promulgated in the final HPV2 test rule. 

There are no outstanding test rule requirements for toxicity testing of CASRN 144-62-7.

 1 The HPV2 test rule specifies that acute fish and daphnid toxicity testing be conducted according to ASTM E729-

96, algae toxicity testing be conducted according to ASTM guideline E 1218-04ε1, 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. 

Massingale (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).
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D. DATA FOR SIDS ENDPOINTS

D-1. Acute Toxicity to Fish

Summary of endpoint: Adequate

Study 1: Acute toxicity study in zebrafish (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 (C2H2O4) from mg of the dihydrate 

(C2H2O4·2H2O) = 0.71 [ratio of molecular weight of ethanedioic acid (90.03 g/mol) to 

ethanedioic 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, Freshwater and Marine” (1996)

Test type: Acute toxicity to fish - static conditions 

GLP compliant?: Yes 

Year study was performed: 2012

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

(self-employed), Veszprém, 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 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.
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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 hours

Concentrationsa 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 measuredb: 0, 4.48, 6.77, 14.62, and 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 measuredb: 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 Range-finding Test (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, 

hydrate (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 static 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), concentrations were measured 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 solutions 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 before 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 100 
Final HPV2 Chemical Data Adequacy Review CASRN 144-62-7

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mg/L nominal concentrations at a pH within the range (6-8.5) recommended by OECD TG 203. 

(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 203) was used as dilution 

water for the range-finding, definitive, 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 

development and validation).” [CiToxLAB 2013a, p. 17] ISO medium had an approximate 

theoretical total hardness of 249 mg CaCO3/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 the 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 Conditions: Static conditions were used “since 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 conducted 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 complementary 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 saturation) 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 Monitoring: 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.
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Statistical Evaluation: EC50 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): Except 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 testing with unadjusted pH (definitive test):

Ethanedioic acid, hydrate (1:2), CASRN 6153-56-6:

96-hour LC50 = 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; corresponding equivalent toxicity values were calculated 

from the values for ethanedioic acid, hydrate (1:2) using a conversion factor of 0.71 (ratio of 

molecular weight of the acid to the dihydrate): 

96-hour LC50 = 39.8 mg/L; 95% confidence interval = 25.4-74.9 mg/L 

96-hour LOEC (mortality 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 LC50 > 32.94 mg/L

96-hour NOEC (mortality or signs of toxicity) = 32.94 mg/L (highest concentration tested)

Ethanedioic acid, CASRN 144-62-7; equivalent toxicity values:

96-hour LC50 > 23.4 mg/L

96-hour NOEC (mortality or signs of toxicity) = 23.4 mg/L (highest concentration tested)

Results remarks: 

Measured Test Concentrations: For the definitive test, nominal 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 was reported at 96 

hours for the highest test concentration (100 mg/L nominal) because all fish had died by 3 hours 

on test. 
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Measured 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 7.32 ± 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 ± 0.251 97 - - 1 At the nominal concentration 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 measured 

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 most 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-ofstudy concentrations. 

For the complementary 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. 
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Cumulative mortality and pH values in the definitive test

Nominal 

Concentration 

(mg/L)

Geometric Mean 

Measured 

Concentration 

(mg/L)

Cumulative 

Mortality at 96 

Hours

pH1

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

100 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 concentrations ≤ 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.” OECD 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 addition 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 adjusted 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 were 
Final HPV2 Chemical Data Adequacy Review CASRN 144-62-7

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observed in this test. During 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 LC50: Since 0% mortality was seen at 100 mg/L 

(nominal) in the complementary test (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% mortality 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 test, and use of measured test 

substance concentrations as the basis for results given that measured test concentrations were not 

maintained within ± 20% of nominal concentrations during the test.

CONCLUSIONS

For ethanedioic acid, hydrate (1:2), the 96-hour LC50 for zebrafish based on mean measured 

concentrations in the definitive test (testing with unadjusted pH) was 56.0 mg/L. The equivalent 

96-hour LC50 for ethanedioic acid (the designated test rule substance) was 39.8 mg/L.

STUDY RELIABILITY

[2] Reliable with restrictions. The study was 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 nominal 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 storage 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 LC50 values could be affected by the lack of intermediate response levels, 

because the calculation is based on 100% response at the highest concentration and 0% response 

at lower levels and in controls (essentially a concentration-response curve with two points).
Final HPV2 Chemical Data Adequacy Review CASRN 144-62-7

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REFERENCES

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 RN 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).
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D-2. Acute Toxicity to Aquatic Invertebrates

Summary of endpoint: Adequate

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

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 (C2H2O4) from mg of the dihydrate 

(C2H2O4·2H2O) = 0.71 [ratio 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 Toxicity Test, Freshwater Daphnids” (1996)

Test type: Daphnia sp., acute immobilization test - static conditions

GLP compliant?: Yes 

Year study was performed: 2012

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

Budapest, Hungary. 

Analytical monitoring: Yes 

 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 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 HPV2 Chemical Data Adequacy Review CASRN 144-62-7

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Exposure duration: 48 hours

Concentrationsa 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 measuredb: 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 measuredb

: 0, 58.26, and 109.4 mg/L

a Concentrations are expressed as mg/L of ethanedioic acid, hydrate (1:2). b Geometric means of initial and 48-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 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 Test (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 beginning 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 solutions at 

the two highest concentrations 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 substance. 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. (After adjustment, the pH 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 [and 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 hardness of 249 mg CaCO3/L.
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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 the 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: Static 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 from the definitive test. In particular, the 

problem with maintaining test concentrations throughout the study suggests 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 testing and “were 

derived from healthy stock (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 culture 

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, oxygen 

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 

determined 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: EC50 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. 
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RESULTS

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 EC50 = 38.89 mg/L; 95% confidence interval = 28.05-61.70 mg/L 

48-hour LOEC = 73.65 mg/L

48-hour NOEC = 21.94 mg/L 

Ethanedioic acid, CASRN 144-62-7; corresponding equivalent toxicity values were calculated 

from the values for ethanedioic acid, hydrate (1:2) using a conversion factor of 0.71 (ratio of 

molecular weight of the acid to the dihydrate):

48-hour EC50 = 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 EC50 > 109.4 mg/L

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

Ethanedioic acid, CASRN 144-62-7; equivalent toxicity values:

48-hour EC50 > 77.7 mg/L

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

Results 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 to the end of exposure.

Measured 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 19
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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. The 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 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): 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 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 Daphnia immobilization and 

pH values observed in the definitive test are shown in the following table. 

Immobilization incidence and pH values observed in the definitive test

Nominal 

Concentration 

(mg/L)

Geometric Mean 

Measured 

Concentration 

(mg/L)

Incidence of 

Immobilization 

at 48 Hours1

Mean pH2

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 20/20 at 100 mg/L vs. 0/20 at all concentrations ≤ 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 (pH 
Final HPV2 Chemical Data Adequacy Review 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.

Immobilization incidence and pH values observed in the complementary test

Nominal 

Concentration 

(mg/L)

Geometric Mean 

Measured 

Concentration 

(mg/L)

Incidence of 

Immobilization 

at 48 Hours1

Mean pH2

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 

adjustment 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 Daphnia 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 EC50: Since 0% immobility was seen at 100 mg/L 

(nominal) in the complementary test (with pH adjustment that yielded test water with a pH of 

~7.7), the 100% immobility at 100 mg/L (nominal) in the definitive test (with unadjusted pH) 

appears to be related to the acidification 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. 
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CONCLUSIONS

For ethanedioic acid, hydrate (1:2), the 48-hour EC50 for Daphnia magna based on mean 

measured concentrations in the definitive test (testing with unadjusted pH) was 38.89 mg/L. The 

equivalent 48-hour EC50 for ethanedioic acid (designated test rule substance) was 27.61 mg/L.

STUDY RELIABILITY

[2] Reliable with restrictions. The study was 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 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 losses were so high in the definitive test, as compared to the 

stability tests. In addition, the reliability of the EC50 values could be affected by the lack of 

intermediate response levels, because the calculation is based on 100% response at the highest 

concentration and 0% response at lower levels and in controls (essentially a concentrationresponse curve with two points). Also, although the EC50 value was reported with 95% 

confidence limits, the slope of the concentration-response curve and its standard error were not 

provided as specified in OECD TG 202. 

REFERENCES

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).
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D-3. Toxicity to Aquatic Plants

Summary of endpoint: Adequate

Study 1: Toxicity to Pseudokirchneriella 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 (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 (C2H2O4) from mg of the dihydrate 

(C2H2O4·2H2O) = 0.71 [ratio of molecular weight of ethanedioic acid (90.03 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, Growth 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 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 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.
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Analytical monitoring: Yes 

Exposure duration: 72 hours

Concentrationsa 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 measuredb: 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 measuredb: 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 cultures 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 the 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 replicates 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 the 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).
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Test Solutions: “Reconstituted algal growth medium (OECD medium, according to OECD 201) 

was used as dilution water for the range finding, definitive 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 medium) 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 definitive and complementary tests, “The algal culture flasks were 

continuously illuminated. The light intensity at the position occupied by algal culture flasks 

during the test was about 8116 lux (equivalent to 110 μE/m2

/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 suitable 

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 culture was determined by [a] 

microscopic method and this cell suspension was diluted with Algal Growth Medium to 107

cells/mL.” [CiToxLAB 2013d, p. 13]

Test Procedure: “The exposure time was 72 hours. The test was started (Day 0) by inoculation 

of a biomass of approximately 104 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 chamber. 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 continuously 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 validated HPLC-UV method as 
Final HPV2 Chemical Data Adequacy Review CASRN 144-62-7

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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 Analysis: 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 ErC50, EbC50 and EyC50 values were determined 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 Bonferroni 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 

(means 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 EC50 (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 EC50 (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 EC50 (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; corresponding equivalent toxicity values were calculated 

from the values for ethanedioic acid, hydrate (1:2) using a conversion factor of 0.71 (ratio of

molecular weight of the acid to the dihydrate):

72-h EC50 (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/L
Final HPV2 Chemical Data Adequacy Review CASRN 144-62-7

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72-h EC50 (yield) = 17.24 mg/L; 95% confidence interval = 16.12-18.81 mg/L

72-h LOEC (yield) = 14.0 mg/L

72-h NOEC (yield) = 5.6 mg/L

72-h EC50 (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 EC50 (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 EC50 (yield) > 34.2 mg/L

72-h LOEC (yield) = 34.2 mg/L

72-h NOEC (yield) < 34.2 mg/L

72-h EC50 (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 EC50 (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 EC50 (yield) > 24.3 mg/L

72-h LOEC (yield) = 24.3 mg/L

72-h NOEC (yield) < 24.3 mg/L

72-h EC50 (biomass) > 24.3 mg/L

72-h LOEC (biomass) = 24.3 mg/L

72-h NOEC (biomass) < 24.3 mg/L

Results 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 13-66% from the initiation of exposure to the end of exposure.
Final HPV2 Chemical Data Adequacy Review CASRN 144-62-7

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Measured 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 7.5 ± 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 ± 2.77 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, respectively. 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.” [CiToxLAB 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 (unadjusted 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-finding 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 

Concentration 

(mg/L)

Geometric 

Mean Measured 

Concentration 

(mg/L)

Mean % Inhibition1

(0-72 hours) Mean pH2

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 8.47
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Nominal 

Concentration 

(mg/L)

Geometric 

Mean Measured 

Concentration 

(mg/L)

Mean % Inhibition1

(0-72 hours) Mean pH2

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 control (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 the 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 complementary 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 % Inhibition1

(0-72 hours) Mean pH2

Growth 

Rate

Biomass 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 water-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. 
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Selection of Data for Derivation of the EC50 Values: Since inhibition of algae growth parameters 

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 50 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 ethanedioic acid also exerts toxicity to algae that is related to molecular 

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 inherent 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 definitive 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

Ethanedioic acid, hydrate (1:2): The 72-hr EC50 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.

Ethanedioic acid (designated test rule substance): The equivalent 72-hr EC50 was 19.29 mg/L for 

growth rate and 17.24 mg/L for yield and biomass. The 72-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 

concentrations 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. 
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REFERENCES

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. 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).
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D-4. Genetic Toxicity – Chromosomal Aberrations

Summary of endpoint: 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 144-62-7])

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 μg of ethanedioic acid (C2H2O4) from μg of the dihydrate 

(C2H2O4·2H2O) = 0.71 [ratio of molecular weight 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; morphology: fibroblast 

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, 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 

does not confirm this identity of the test substance.
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Concentrations tested: 

Preliminary concentration selection 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 buffered 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 excluding 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 ensure 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 diluting 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 adjusted 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 treatment) ethyl methanesulfonate (EMS) in Dulbecco’s Modified Eagle’s Medium 

 1 The study 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 HPV2 Chemical Data Adequacy Review CASRN 144-62-7

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(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 cytotoxicity test were used to determine treatment 

concentrations for the main study. The preliminary test was conducted as two separate assays in 

which concentrations 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. Single 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 concentration 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 concentrations

(based on observed solubility and/or cytotoxicity results) from each assay were selected for 

chromosomal aberration analysis. At least 200 well-spread metaphase cells (from both duplicate 

cultures combined) were analyzed (or until a clear positive response was detected) for each 

selected test concentration and negative and positive controls. 

“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 

recorded.

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 concentrations.
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• The increases were reproducible between replicate cultures and between tests with 

identical treatment conditions.

• The increases were statistically significant.

• The increases were not associated with large changes in pH or osmolality in the treated 

cultures. (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 metabolic 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 test 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 concentrations 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 

statistically 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. 

Chromosome 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 test 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/mL tested with S9. 
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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 control 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 samples 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 historical 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. Results 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 groups and 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 

cells 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 presented 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 regarding the ability of ethanedioic acid to induce chromosomal aberrations 

in mammalian cells in vitro. 

 
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REFERENCES

CiToxLAB Hungary Ltd. 2013e. In vitro Mammalian Chromosome Aberration Test of 

Ethanedioic acid, hydrate (1:2) [CAS RN 6153-56-6] (hydrated form of ethanedioic acid [CAS 

RN 144-62-7]). Study code: 11/339-020C. Study Director: Judit Hargitai. 22 March 2013.

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