SRC TR-13-016

HPV1/HPV2 TEST RULE DATA ADEQUACY REVIEW: MAY 2013 UPDATE (DRAFT)

9,10-Anthracenedione, CASRN 84-65-1

Prepared by:

William L. Richards, Ashlee Aldridge, Justine Von Runnen, Teresa Manyin, Kelly Salinas, 

Christopher Janson, Lisa Ingerman, Jennifer Rayner, and Julie Klotzbach

Defense and Environmental Solutions Division

SRC, Inc.

7502 Round Pond Road

North Syracuse, NY 13212

Contract No. EP-W-09-027

Task FG007.3.009.004

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

May 31, 2013
Draft HPV1/HPV2 Chemical Data Adequacy Review (May 2013) CASRN 84-65-1

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TABLE OF CONTENTS

A. SOURCE MATERIAL USED FOR ASSESSMENT .............................................................. 1

B. EVALUATING DATA ADEQUACY ..................................................................................... 3

C. DATA FOR SIDS ENDPOINTS .............................................................................................. 4

C-1. Acute Toxicity to Fish........................................................................................................ 4

C-2. Acute Toxicity to Aquatic Invertebrates.......................................................................... 29

C-3. Toxicity to Aquatic Plants................................................................................................ 40

C-4. Acute Toxicity.................................................................................................................. 47

C-5. Repeated-Dose Toxicity................................................................................................... 52

C-6. Genetic Toxicity – Gene Mutation................................................................................... 74

C-7. Genetic Toxicity – Chromosomal Aberrations................................................................ 84

C-8. Reproductive/Developmental Toxicity ............................................................................ 95

D. SUPPLEMENTAL DATA (NON-SIDS ENDPOINTS)...................................................... 102

D-1. Skin Irritation ................................................................................................................. 102

D-2. Eye Irritation .................................................................................................................. 108

D-3. Skin Sensitization........................................................................................................... 114

D-4. Genetic Toxicity – Other................................................................................................ 118

E. DATA ADEQUACY SUMMARY TABLE......................................................................... 120
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A. SOURCE MATERIAL USED FOR ASSESSMENT

In this document, toxicological data for 9,10-anthracenedione (CASRN 84-65-1) are summarized 

and evaluated for data adequacy. This report provides an update of the data adequacy 

assessment that was conducted previously in the following report: “HPV1/HPV2 Test Rule Data 

Adequacy Review: November 2012 Update (draft): 9,10-Anthracenedione, CASRN 84-65-1. 

SRC, Inc., Technical Report #12-085. November 8, 2012.” 

In HPV TSCA Section 4 test rules, CASRN 84-65-1 is subject to toxicological testing 

requirements consisting of the following: (1) F2 (reproduction/developmental toxicity screening 

test following 40 CFR 799.9355) in the first HPV TSCA Section 4 Test Rule (Testing of Certain 

High Production Volume Chemicals; Final Rule, 71 FR 13708, March 16, 2006; 

http://www.regulations.gov docket EPA-HQ-OPPT-2005-0033; referred to herein as HPV1); and 

(2) C6 (Toxicity to Plants [Algae] testing following ASTM guideline E 1218-04e1) in the second 

HPV TSCA Section 4 test rule (Testing of Certain High Production Volume Chemicals; Second 

Group of Chemicals; Final Rule; 76 FR 1067, January 7, 2011; http://www.regulations.gov

docket EPA-HQ-OPPT-2007-0531; referred to herein as HPV2).

As compared to the November 2012 data adequacy assessment for CASRN 84-65-1, this current

updated document includes additional information concerning aquatic toxicity. Major revisions 

include: (1) addition of details to aquatic toxicity studies for which the study report or primary 

source was located, (2) addition of an algal toxicity study on an analogous chemical (1,4-

anthraquinone)1, and (3) revision of the status of the testing requirement for toxicity to plants 

(algae). Details of these revisions follow.

(1) This document includes summaries of studies that were conducted in response to EPA’s 

1987 test rule for CASRN 84-65-1 (Anthraquinone; Final Reporting and Recordkeeping 

Requirements and Test Rule; 52 FR 21018, June 4, 1987); these five aquatic toxicity studies 

are cited as Springborn Life Sciences, Inc., 1988a-e. These studies were evaluated by EPA 

at the time of submission and the data were found to be unreliable and not adequate for risk 

assessment or other purposes. Although the previous (November 2012) draft of the current 

document presented these studies as “adequate” based on information available from 

European Chemicals Agency (ECHA) website (http://echa.europa.eu/), additional 

information acquired from EPA’s archives, including the original study reports and EPA’s 

original reviews, has revealed that these studies are not adequate. The deficiencies of each 

study are elaborated in Section C of this document. 

(2) An algal toxicity study by Giddings (1979)1 was identified and assessed for data 

adequacy. This study was conducted using an analogue (1,4-anthraquinone) of CASRN 84-

65-1. The study was determined to be inadequate to satisfy the C6 testing requirement due 

to its brief exposure duration, lack of methodological details, and absence of analytical 

measurement of test substance concentrations.

 

1 Giddings. J.M. 1979. Acute toxicity to Selenastrum capricornutum of aromatic compounds from coal conversion. 

Bull. Environm. Contam. Toxicol. 23: 360-364.
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(3) Chemical Products Corporation requested in a March 16, 2012 email that EPA accept 

preexisting data from Chillingworth (1974)1 to satisfy the C6 testing requirement for 

9,10-anthracenedione. Although these data are not considered adequate to satisfy the C6 

testing requirement, as explained in Section C of this document, the toxicity to aquatic 

plants endpoint is not considered to be a data gap at this time, given that: (1) low toxicity 

has been observed in fish and daphnids (i.e., no adverse effects at or near the limit of water 

solubility); and (2) according to EPA’s 1987 test rule for CASRN 84-65-1 (Anthraquinone; 

Final Reporting and Recordkeeping Requirements and Test Rule; 52 FR 21018, June 4, 

1987), “sufficient data are available from the study done by Chillingworth [1974]... to 

reasonably predict anthraquinone’s toxicity to algae”. Therefore, there is no need for further 

testing of the toxicity to aquatic plants endpoint at this time. 

This document also includes data submitted in response to the F2 testing requirement in the 

HPV1 test rule. EPA received the following study, which was considered adequate to satisfy the 

F2 testing requirement, on May 7, 2012 from Chemical Products Corporation, the sponsor for 

9,10-anthracenedione:

MPI Research, Inc. 2012. An Oral Reproduction/Developmental Toxicity Screening Study 

of Anthraquinone (CAS #84-65-1) in Rats. Study No. 1271-001. Date of Study 

Completion: February 6, 2007. Revised Report Dated May 4, 2012. Available at: 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2005-0033-0413 as of 

November 8, 2012.

Data for the first (F2) and second (C6) HPV test rule-required endpoints, as well as available 

data for other SIDS toxicity endpoints, are evaluated for adequacy in Section C. Additional data 

for non-SIDS toxicity endpoints (“supplemental” data) are reviewed in Section D. Sections C 

and D incorporate information from the High Production Volume Information System (HPVIS) 

and the European Chemicals Agency (ECHA) website (http://echa.europa.eu/). A summary of 

the adequacy assessment for each SIDS endpoint is provided on the last page of this document 

(Section E). 

 

1 Chillingworth, M.A. 1974. The toxicity of aminoanthraquinone dyes to fish and algae. M.S.P.H. Thesis, Dept. 

Environ. Sci. Eng., Univ. North Carolina, Chapel Hill.
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B. EVALUATING DATA ADEQUACY

Each study was summarized according to a standard format and assigned a Klimisch reliability 

code based on its scientific merit and conduct, using professional judgment. To determine 

whether data were adequate to satisfy SIDS endpoints, study details were compared to current 

standard test guidelines to establish whether the study met current testing requirements. Studies 

for a common endpoint are grouped together in the document and an endpoint adequacy 

assessment is provided near the top of the first page of the summaries for that endpoint. If the 

data available for the endpoint were determined to be inadequate, a basis for that conclusion is 

supplied. A table summarizing data availability/adequacy for each SIDS endpoint is included at 

the end of the summary document. 
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C. DATA FOR SIDS ENDPOINTS

C-1. Acute Toxicity to Fish

Summary of endpoint: Adequate

Study 1: Acute toxicity study in bluegill (EPA, 2006)

Title: Anthraquinone (122701) Fact Sheet, Biopesticides and Pollution Prevention Division 

Office of Pesticide Programs

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: 9,10-Anthracenedione, CASRN 84-65-1

Remarks: Purity not provided; test substance identified as anthraquinone

METHOD

Method/guideline followed: No data

Test type: No data

GLP compliant?: No data

Year study was performed: No data

Test species: Bluegill sunfish (Lepomis macrochirus)

Analytical monitoring: No data

Exposure duration: 96 hours

Concentrations tested: No data

Test method/conditions remarks: No data

RESULTS

Endpoint value(s): 96-hour LC50 > 190 μg/L; NOEC > 190 μg/L

Results remarks: The Office of Pesticide Programs “Anthraquinone (122701) Fact Sheet” 

reports the LC50 and NOEC in units of “Fg/L”, i.e., LC50 > 190 Fg/L and NOEC > 190 Fg/L. 

The meaning of “Fg” is not defined in the Fact Sheet. Based on the solubility limit of CASRN 
Draft HPV1/HPV2 Chemical Data Adequacy Review (May 2013) CASRN 84-65-1

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84-65-1, the units are assumed to be μg/L; the use of “Fg/L” rather than μg/L appears to be a 

transcription error. The original study and the Data Evaluation Review for this study were not 

available for review; the study report is considered to be FIFRA proprietary information.

CONCLUSIONS

The 96-hour LC50 for Lepomis macrochirus was > 190 μg/L.

STUDY RELIABILITY

[1] Reliable without restrictions. This study, MRID # 44496122, is considered acceptable by 

OPP.

REFERENCES

EPA. 2006. Anthraquinone (122701) Fact Sheet, Biopesticides and Pollution Prevention 

Division Office of Pesticide Programs, EPA, Washington, DC. Available at 

http://www.epa.gov/pesticides/chem_search/reg_actions/registration/fs_PC-122701_01-Dec98.pdf as of May 22, 2013.
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Study 2: Acute Toxicity to Oncorhynchus mykiss (EPA, 2006)

Title: Anthraquinone (122701) Fact Sheet, Biopesticides and Pollution Prevention Division 

Office of Pesticide Programs

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: 9,10-Anthracenedione, CASRN 84-65-1

Remarks: Purity not provided; test substance identified as anthraquinone

METHOD

Method/guideline followed: No data

Test type: No data

GLP compliant?: No data

Year study was performed: No data

Test species: Rainbow trout (Oncorhynchus mykiss)

Analytical monitoring: No data

Exposure duration: 96 hours

Concentrations tested: No data

Test method/conditions remarks: No data

RESULTS

Endpoint value(s): 96-hour LC50 > 150 μg/L; NOEC > 150 μg/L

Results remarks: The Office of Pesticide Programs “Anthraquinone (122701) Fact Sheet” 

reports the LC50 and NOEC in units of “Fg/L”, i.e., LC50 > 150 Fg/L and NOEC > 150 Fg/L.The 

meaning of “Fg” is not defined in the Fact Sheet. Based on the solubility limit of CASRN 84-

65-1, the units are assumed to be μg/L; the use of “Fg/L” rather than μg/L appears to be a 

transcription error. The original study and the Data Evaluation Review for this study were not 

available for review; the study report is considered to be FIFRA proprietary information.
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CONCLUSIONS

The 96-hour LC50 for Oncorhynchus mykiss was > 150 μg/L.

STUDY RELIABILITY

[1] Reliable without restrictions. This study, MRID # 44496123, is considered acceptable by 

OPP.

REFERENCES

EPA. 2006. Anthraquinone (122701) Fact Sheet, Biopesticides and Pollution Prevention 

Division Office of Pesticide Programs, EPA, Washington, DC. Available at 

http://www.epa.gov/pesticides/chem_search/reg_actions/registration/fs_PC-122701_01-Dec98.pdf as of May 22, 2013.
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Study 3: Acute toxicity study in rainbow trout (Springborn Life Sciences, Inc., 1988a) 

Title: Acute Toxicity of Anthraquinone to Rainbow Trout (Salmo gairdneri) under FlowThrough Conditions

This study is: Adequate Not Adequate

The study had the following deficiencies compared with the EPA 797.1400 test guideline: (1) the 

measured test concentrations at 0 hours in replicates A and B at nominal concentrations of 65 

and 18 μg/L varied by more than the maximum of 20 percent allowed by the guideline; (2) 

between 0 hours and 96 hours, the measured test concentration at the nominal concentrations of 

18 and 42 μg/L varied by more than the maximum of 30% allowed by the guideline; (3) the 

dissolved oxygen levels in 7/14 replicates at 96 hours were ≤ 8.2 mg/L, whereas the guideline 

specifies dissolved oxygen shall be maintained above 8.2 mg/L in flow-through tests with trout; 

(4) the test temperature varied from the guideline-specified range of 12 ± 2°C (range during test: 

13-15°C); (5) the acidity, total organic carbon or chemical oxygen demand, and particulate 

matter of the dilution water were not reported; (6) the water solubility determinations and 

adequacy of the methods to attain maximum test concentrations were not adequately explained; 

(7) maximum water solubility at the highest test concentration was not achieved based on a 

validated water solubility study conducted after the toxicity test, i.e., the highest test 

concentration of 55 μg/L was approximately 27% lower than the measured maximum water 

solubility of 76.1 μg/L at a temperature and pH closely approximating the fish test conditions (12 

°C and pH of 7.0); (8) the age of the fish was not reported; (9) the concentration of acetone used 

(75 μL/L) exceeded the test guideline maximum of 5 mg/L (equivalent to ~ 6.2 μL acetone/L); 

(10) the report did not specify the replicate in which sublethal effects were observed at 48 hours; 

and (11) the report did not indicate which pesticides were measured analytically or provide data 

on other possible contaminants in the dilution water.

TEST SUBSTANCE 

Identity: 9,10-Anthracenedione, CASRN 84-65-1 

Remarks: Purity: 99.81% active ingredient (A.I.) according to the study sponsor; 50:50 

mixture of Mobay and ICI anthraquinone powders (Mobay lot #2025865; ICI lot# 611884); 

yellow powder received by the Mobay Corporation.

METHOD

Method/guideline followed: TSCA Guidelines (number of guideline not specified)

Test type: Acute toxicity to fish (flow-through)

GLP compliant?: Yes

Year study was performed: 1988
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Test species: Rainbow trout (Oncorhynchus mykiss)

Analytical monitoring: Yes; HPLC

Exposure duration: 96 hours 

Concentrations tested: Nominal concentrations: 0, 18, 27, 42, 65, and 100 μg a.i./L; Mean 

measured concentrations: 0, 10, 18, 23, 35, and 55 μg a.i./L.

Test method/conditions remarks: “The test was conducted using an exposure system 

consisting of a constant flow serial diluter..., a temperature-controlled water bath, and set of 14 

exposure aquaria... [Twenty organisms were exposed in duplicate test aquaria (10 fish per 

replicate) in a flow-through system] to five concentrations of the test substance, one dilution 

water control, and one solvent control. The solvent control solutions contained the maximum 

amount of acetone... present in any test concentration (75 μL/L). All treatment levels and the 

controls were maintained in duplicate. Test aquaria were labeled to identify the test sample, 

nominal test concentration, and designated replicate. Test solutions were not aerated. A 

photoperiod of 16 hours light and 8 hours darkness with a light intensity of 30-60 footcandles at 

the test solution surface was maintained throughout the test period.” [Springborn Life Sciences, 

Inc. 1988a, p. 8-9]

The dilution water was from the same source as the water that flowed into the fish holding tank. 

“Each glass test aquarium measured 39 x 20 x 25 centimeters (cm) with a 19.5 cm high 

standpipe, which maintained a constant test water volume of 15 L. The diluter was constructed 

to deliver 68 mL of solution per minute to each replicate test aquarium, which provided 

approximately 6.5 volume replacements per aquarium every 24 hours. Test aquaria were 

impartially positioned in a water bath containing circulating water cooled by a Frigid Unit 

Chiller designed to maintain the test solution temperatures at 12 ± 2°C.” [Springborn Life 

Sciences, Inc. 1988a, p. 10] 

“The test was initiated when ten rainbow trout were impartially selected and distributed to each 

replicate aquarium. At any given time during the test, the maximum organism loading 

concentration was 0.15 g of biomass per L of flowing test solution per day.” [Springborn Life 

Sciences, Inc. 1988a, p. 11]

Biological observations were made and recorded at test initiation and every 24 hours thereafter 

until the test was terminated. 

“Selection of the nominal concentrations for this study was based on the limit of water solubility 

of anthraquinone in freshwater under the maintained test conditions. Prior to initiation of the 

definitive study, preliminary testing was conducted at Springborn Life Sciences, Inc. (SLS) to 

establish the water solubility and acute toxicity of anthraquinone under test conditions. Test 

conditions for this study were generally consistent with the requirements published in the EPA's 

Final Test Rule, Parts 704, 795, and 799 (Federal Register Volume 52, No. 107, 4 June 1987). 

Preliminary testing corroborated information provided by the Mobay Corporation and 

established that the limit of water solubility for anthraquinone, under the maintained test 

conditions, was approximately 50 μg/L.” [Springborn Life Sciences, Inc. 1988a, p. 14]
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Details on Sampling: “All test concentrations and the control solution were sampled and 

analyzed for anthraquinone concentrations prior to the start of the definitive exposure. Results of 

these pretest analyses were used to judge whether sufficient quantities of test material were being 

delivered and maintained in the exposure aquaria to initiate the definitive test. During the in-life 

phase of the definitive study, water samples were removed from both replicate test solutions of 

each treatment level and the controls on test days 0 and 4 for analysis of anthraquinone. Each 

exposure solution sample was collected from the approximate midpoint of the aquarium with a 

volumetric pipet. In addition, quality assurance (QA) blind samples were prepared at each 

sampling interval and remained with the set of exposure solution samples through the analytical 

process. These QA samples were prepared in dilution water at anthraquinone concentrations 

unknown to the analyst. Results of the analyses of the QA samples were used to judge the 

precision and quality control maintained during the analysis of exposure solution samples.” 

[Springborn Life Sciences, Inc. 1988a, p. 12-13]

Details on Test Solutions: “A diluter stock solution of 1.34 mg A.I./mL was prepared by diluting 

0.3358 g of Anthraquinone (0.3352 grams active ingredient [A.I]) with acetone to a total volume 

of 250 mL. A Sage syringe pump in conjunction with a 50-mL gas-tight syringe was used to 

deliver the stock solution of Anthraquinone (1.34 mg A.I./mL) to the diluter's chemical mixing 

chamber containing approximately 1 L of dilution water. The mixing chamber was positioned 

over a magnetic stirrer and inside of an ultrasonic water bath. The continuous stirring and 

sonication aided in the solubilization of the test material. The solution contained in the mixing 

chamber constituted the highest nominal test concentration (100 μg A.I./L) and was subsequently 

diluted (65% dilution factor) to provide the desired exposure concentration range. A system 

similar to that used to deliver the test material was used to deliver acetone to the solvent control 

aquaria.” [Springborn Life Sciences, Inc. 1988a, p. 9-10]

 

Details on Test Organisms: “The rainbow trout (SLS lot #88A30) were obtained from a 

commercial supplier in Montana. Prior to testing, these fish were held in 500-L fiberglass 

aquaria, under a photoperiod of 16 hours light and 8 hours darkness. The well water that flowed 

into this holding tank was characterized as having a total hardness and alkalinity range as 

calcium carbonate (CaCO3) of 24-28 and 19-22 mg/L, respectively, and a specific conductance 

range of 100-130 micromhos per centimeter (μmhos/cm) (Weekly Gravity Feed Tank Water 

Quality Analysis Log Book). Other parameters monitored in the holding tank were a pH range 

of 6.9-7.1, a dissolved oxygen concentration range of 76-83% of saturation, and a flow rate of 

8.8-11.5 tank volume replacements/day (Weekly Record of Fish Holding Characteristics). Test 

fish were maintained under similar conditions for a minimum of 14 days prior to testing. The 

temperature range in the holding tank was 13-14°C during this 14-day period. All fish were fed 

a dry commercial pelleted food, ad libitum, daily. There was 0.4% mortality in the test fish 

population during the two days prior to testing. The rainbow trout used during this study were 

all of the same year class and a representative sample (n=30) of the fish from this population had 

a mean (range) wet weight of 1.49 (0.55-2.43) g and total length of 55 (41-65) mm (Fish Weight 

and Lengths Log).” [Springborn Life Sciences, Inc. 1988a, p. 7-8].

Protocol deviations were reported and included low dissolved oxygen (dropped below 8.2 mg/L; 

range during test was 7.3-10.0 mg/L [69-95% of saturation]), high temperature of test solutions
Draft HPV1/HPV2 Chemical Data Adequacy Review (May 2013) CASRN 84-65-1

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(above 14°C; range during test was 13-15°C), and type of diluter used (flow serial diluter was 

used instead of a proportional diluter). 

RESULTS

Endpoint value(s): 96-hour LC50 > 55 μg a.i./L (based on mean measured concentrations)

Results remarks: “Throughout the exposure period, a small amount of precipitated test material

was observed in the diluter system's mixing chamber, however, no undissolved Anthraquinone

(e.g., precipitate, film on solution's surface) was observed in any of the exposure vessels during 

the study.” [Springborn Life Sciences, Inc. 1988a, p. 15]

At some treatment levels, measured concentrations of test substance varied by more than 30%

during the 96-hour test period. At the nominal concentration of 42 μg/L, the measured 

concentration of one replicate increased by 44% from 0 to 96 hours. At the nominal 

concentration of 18 μg/L, the measured concentration of one replicate increased by 48% from 0 

to 96 hours, while the measured concentration of the other replicate increased by > 103%. The 

measured concentrations of all other replicates varied by less than 30% during the period of 

exposure. 

Measured test concentrations at nominal concentrations of 18 and 65 μg/L varied more than 20% 

between replicates at 0 hours. At 18 μg/L (nominal), the difference between replicates was > 

50% at 0 hours. At 65 μg/L (nominal), the difference between replicates was 31% at 0 hours. 

During 96 hours of exposure, one fish died at 55 μg a.i./L and two fish died at 23 μg a.i./L, 

resulting in mortality rates of 5% and 10% at 55 and 23 μg a.i./L, respectively. No mortalities 

were observed at other test concentrations. All deaths were observed at the 96-hour observation 

period. Loss of equilibrium was observed in one fish at 23 μg a.i./L at 48 hours. The study 

authors did not consider the mortalities and sublethal effects to be toxicant-related.

During the 96-hour exposure period, pH ranged from 6.5 to 7.2, dissolved oxygen ranged from 

7.3 to 10.0 mg/L, and temperature ranged from 13 to 15°C.

CONCLUSIONS

The 96-hour EC50 for rainbow trout was > 55 μg a.i./L.

“Based on these data it was established that under the test conditions maintained during this 

study (e.g., water quality, solution temperature, solvent concentration, mixing period) 

Anthraquinone was not acutely toxic to rainbow trout at the material's limit of water solubility.” 

[Springborn Life Sciences, Inc. 1988a, p. 17]
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STUDY RELIABILITY

[3] Not reliable. This study was submitted in response to EPA’s 1987 test rule for CASRN 84-

65-1 (Anthraquinone; Final Reporting and Recordkeeping Requirements and Test Rule; 52 FR 

21018, June 4, 1987) and was found to have the following deficiencies compared with the EPA 

797.1400 test guideline: (1) the measured test concentrations at 0 hours in replicates A and B at

nominal concentrations of 65 and 18 μg/L varied by more than the maximum of 20 percent

allowed by the guideline; (2) between 0 hours and 96 hours, the measured test concentration at 

the nominal concentrations of 18 and 42 μg/L varied by more than the maximum of 30% allowed 

by the guideline; (3) the dissolved oxygen levels in 7/14 replicates at 96 hours were ≤ 8.2 mg/L, 

whereas the guideline specifies dissolved oxygen shall be maintained above 8.2 mg/L in flowthrough tests with trout; (4) the test temperature varied from the guideline-specified range of 12 

± 2°C (range during test: 13-15°C); (5) the acidity, total organic carbon or chemical oxygen 

demand, and particulate matter of the dilution water were not reported; (6) the water solubility 

determinations and adequacy of the methods to attain maximum test concentrations were not 

adequately explained; (7) maximum water solubility at the highest test concentration was not 

achieved based on a validated water solubility study conducted after the toxicity test, i.e., the 

highest test concentration of 55 μg/L was approximately 27% lower than the measured 

maximum water solubility of 76.1 μg/L at a temperature and pH closely approximating the fish 

test conditions (12 °C and pH of 7.0); (8) the age of the fish was not reported; (9) the 

concentration of acetone used (75 μL/L) exceeded the test guideline maximum of 5 mg/L 

(equivalent to ~ 6.2 μL acetone/L); (10) the report did not specify the replicate in which 

sublethal effects were observed at 48 hours; and (11) the report did not indicate which pesticides 

were measured analytically or provide data on other possible contaminants in the dilution water. 

REFERENCES

Springborn Life Sciences, Inc. 1988a. Acute Toxicity of Anthraquinone to Rainbow Trout

(Salmo gairdneri) under Flow-Through Conditions. Report # 88-10-2831. SLS Study 

#274.0488.6176.108. Sponsor: Mobay Corporation. 

Text in quotes is taken directly from Springborn Life Sciences, Inc., 1988a.
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Study 4: Acute toxicity study in coho salmon (Springborn Life Sciences, Inc., 1988b) 

Title: Acute Toxicity of Anthraquinone to Coho Salmon (Orcorhynchus kisutch) under FlowThrough Conditions

This study is: Adequate Not Adequate

The study had the following deficiencies compared with the EPA 797.1400 test guideline: (1) the 

age of the fish was not reported; (2) the acidity, total organic carbon or chemical oxygen 

demand, and particulate matter of the dilution water were not reported; (3) the report did not 

indicate which pesticides were measured analytically or provide data on other possible 

contaminants in the dilution water; (4) the test temperature varied from the guideline-specifed 

range of 12 ± 2°C for cold-water species (range during test: 13-15°C); (5) 18 measurements of 

dissolved oxygen concentrations were less than 8.2 mg/L, whereas the guideline specifies 

dissolved oxygen shall be maintained above 8.2 mg/L in flow-through tests with cold-water 

species such as trout; (6) at 0 hours, there was a relatively large difference in pH levels (up to 0.6 

pH units) between replicates for most groups, including the controls; (7) the measured test 

concentrations at 96 hours in replicates A and B at nominal concentrations of 100, 27 and 18 

μg/L varied by more than the maximum of 20% allowed by the guideline; (8) between 0 hours 

and 96 hours, the measured test concentration at the nominal concentration of 18 μg/L varied by 

more than the maximum of 30% allowed by the guideline; (9) the concentration of acetone used 

(75 μL/L) exceeded the test guideline maximum of 5 mg/L (equivalent to ~ 6.2 μL acetone/L); 

(10) the water solubility determinations and adequacy of the methods to attain maximum test 

concentrations were not adequately explained; and (11) the maximum water solubility at the 

highest test concentration was not achieved based on a validated water solubility study 

conducted after the toxicity test, i.e., the highest test concentration of 45 μg/L was ~59% of the 

measured maximum water solubility of 76.1 μg/L at a temperature and pH closely approximating 

the fish test conditions (12 °C and pH of 7.0). 

TEST SUBSTANCE 

Identity: 9,10-Anthracenedione, CASRN 84-65-1 

Remarks: Purity: 99.81% active ingredient (A.I.) according to the study sponsor; 50:50 

mixture of Mobay and ICI anthraquinone powders (Mobay lot #2025865; ICI lot# 611884); 

yellow powder received by the Mobay Corporation.

METHOD

Method/guideline followed: TSCA Guidelines (number of guideline not specified)

Test type: Acute toxicity to fish (flow-through)

GLP compliant?: Yes

Year study was performed: 1988
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Test species: Coho salmon (Oncorhynchus kisutch)

Analytical monitoring: Yes; HPLC

Exposure duration: 96 hours 

Concentrations tested: Nominal concentrations: 0, 18, 27, 42, 65, and 100 μg a.i./L; Mean 

measured concentrations: 0, 12, 18, 24, 30, and 45 μg a.i./L.

Test method/conditions remarks: “The test was conducted using an exposure system 

consisting of a constant flow serial diluter..., a temperature controlled water bath, and a set of 14 

exposure aquaria... [Twenty organisms were exposed in duplicate test aquaria (10 fish per 

replicate) in a flow-through system] to five concentrations of the test substance, a dilution water 

control, and a solvent control. The solvent control solutions contained the maximum amount of 

acetone... present in any test concentration (75 μL/L). All treatment levels and the controls were

maintained in duplicate. Test aquaria were labeled to identify the test sample, nominal test 

concentration, and designated replicate. Test solutions were not aerated. A photoperiod of 16 

hours light and 8 hours darkness with a light intensity or 30-60 footcandles at the test solution 

surface was maintained throughout the test period. Lighting was provided by Vita-LiteR 

fluorescent bulbs.” [Springborn Life Sciences, Inc. 1988b, p. 8-9]

“Each glass test aquarium measured 39 x 20 x 25 centimeters (cm) with a 19.5 cm high 

standpipe, which maintained a constant test water volume of 15 L. The diluter was constructed 

to deliver 68 mL of solution per minute to each replicate test aquarium, which provided 

approximately 6.5 volume replacements per aquarium every 24 hours. Test aquaria were 

impartially positioned in a water bath containing circulating water cooled by a Frigid Unit 

Chiller designed to maintain the test solution temperatures at 12 ± 2°C.” [Springborn Life 

Sciences, Inc. 1988b, p. 10] 

“Selection of the nominal concentrations for this study was based on the limit of water solubility 

of anthraquinone in freshwater under the maintained test conditions. Prior to initiation of the 

definitive study, preliminary testing was conducted at Springborn Life Sciences, Inc. (SLS) to 

establish the water solubility and acute toxicity of anthraquinone under test conditions. Test 

conditions for this study were generally consistent with the requirements published in the EPA's 

Final Test Rule, Parts 704, 795, and 799 (Federal Register Volume 52, No. 107, 4 June 1987). 

Preliminary testing corroborated information provided by the Mobay Corporation and 

established that the limit of water solubility for the test substance, under the maintained test 

conditions, was approximately 50 μg/L. During this preliminary test period, it was also 

determined that concentrations of ≤ 50 μg/L of test substance were not acutely toxic to coho 

salmon.” [Springborn Life Sciences, Inc. 1988b, p. 14]

Test Conditions throughout the study included: Test Temperature: 13-15°C; pH: 6.5-7.6; 

Dissolved oxygen: 7.3-9.9 mg/L (71-96% saturation).

Details on Test Solutions: “A diluter stock solution of 1.34 mg A.I./mL was prepared by diluting 

0.3358 g of Anthraquinone (0.3352 grams of active ingredient) with acetone to a total volume of 
Draft HPV1/HPV2 Chemical Data Adequacy Review (May 2013) CASRN 84-65-1

15

250 mL. A Sage syringe pump in conjunction with a 50-mL gas-tight syringe was used to 

deliver the stock solution of Anthraquinone (1.34 mg A.I./mL) to the diluter's chemical mixing 

chamber containing approximately 1 L of dilution water. The mixing chamber was positioned 

over a magnetic stirrer and inside of an ultrasonic water bath. The continuous stirring and 

sonication aided in the solubilization to the test material. The solution contained in the mixing 

chamber constituted the highest nominal test concentration (100 μg A.I./L) and was subsequently 

diluted (65% dilution factor) to provide the desired exposure concentration range. A system 

similar to that used to deliver the test material was used to deliver acetone to the solvent control 

aquaria.” [Springborn Life Sciences, Inc. 1988b, p. 9-10]

Details on Test Organisms: “The coho salmon (SLS lot #88A28) were obtained from a 

commercial supplier in Washington. Prior to testing, these fish were held in a 500-L fiberglass 

tank under a photoperiod of 16 hours light and 8 hours darkness. The well water that flowed into 

this holding tank was characterized as having a total hardness and alkalinity range as calcium 

carbonate (CaCO3) of 24-28 mg/L and 19-22 mg/L, respectively, and a specific conductance 

range of 100-130 micromhos per centimeter (μmhos/cm) (Weekly Gravity Feed Tank Water 

Quality Analysis Log Book). Other parameters monitored in the holding tank were a pH range 

of 7.0-7.3, a dissolved oxygen concentration range of 68-83% of saturation and a flow rate of 

9.6-13 tank volume replacements/day... Test fish were maintained under similar conditions for a 

minimum of 14 days prior to testing. The temperature range in the holding tank was 11-14°C 

during this 14-day period. All fish were fed a dry commercial pelleted food, ad libitum, daily. 

There was 0.10% mortality in the test fish population during the 2 days prior to testing... The 

coho salmon used during this study were all of the same year class and a representative sample 

(n=30) of the fish from this population had a mean (range) wet weight of 1.75 (0.63-3.21) grams

and total length of 59 (44-73) millimeters...” [Springborn Life Sciences, Inc. 1988b, p. 7-8]

“The test was initiated when ten coho salmon were impartially selected and distributed to each 

replicate aquarium. At any given time during the test, the maximum organism loading 

concentration was 0.18 g of biomass per L of flowing test solution per day.” [Springborn Life 

Sciences, Inc. 1988b, p. 11]

Each replicate solution was sampled and analyzed for the test substance concentration at test 

initiation and on day 4 of the exposure period. Biological observations were made and recorded 

at test initiation and every 24 hours thereafter until the test was terminated.

The following protocol deviations were reported: fish were fed during the final 48 hours before 

the test was initiated, total alkalinity was 19-22 mg/L CaCO3 instead of 20-35 mg/L CaCO3, 

dissolved oxygen was 7.3-9.9 mg/L (71-96% of saturation) which was below the 8.2 mg/L limit,

temperature was 13-15°C which was outside the specified range of 12 ± 2°C, and a flow serial 

diluter was used instead of a proportional diluter.

RESULTS

Endpoint values: 96-hour LC50 > 45 μg a.i./L (based on mean measured concentrations); 

NOEC = 45 μg a.i./L (based on mean measured concentrations)
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Results remarks: “Throughout the exposure period, a small amount of precipitated test material

was observed in the diluter system's mixing chamber, however no undissolved Anthraquinone 

(e.g., precipitate, film on solution's surface) was observed in any of the exposure vessels during 

the study.” [Springborn Life Sciences, Inc. 1988b, p. 15]

Measured concentrations of test substance decreased during the 96-hour test period. The 

measured concentration decreased by 471, 301, 28-29, 24-26, and 18-38% at nominal 

concentrations of 18, 27, 42, 65, and 100 μg/L, respectively, from 0 to 96 hours. Measured test 

concentrations at nominal concentrations of 18, 27, and 100 μg/L varied more than 20% between 

replicates at 96 hours. At 100 μg/L (nominal), the difference between replicates was 21% at 96 

hours. At 27 μg/L (nominal), the difference between replicates was greater than 61% at 96 

hours. At 18 μg/L (nominal), the difference between replicates was greater than 31% at 96 

hours.

During 96 hours of exposure, no mortalities or adverse effects were observed among coho 

salmon exposed to any of the tested concentrations (12-45 μg a.i./L).

CONCLUSIONS

The 96-hour EC50 for coho salmon was > 45 μg a.i. /L; 96-hour NOEC = 45 μg a.i./L.

“Based on these data it was established that under the test conditions maintained during this 

study (e.g., water quality, solution temperature, solvent concentration, mixing period) 

Anthraquinone was not acutely toxic to coho salmon at the material's limit of water solubility.” 

[Springborn Life Sciences, Inc. 1988b, p. 17]

STUDY RELIABILITY

[3] Not reliable. This study was submitted in response to EPA’s 1987 test rule for CASRN 84-

65-1 (Anthraquinone; Final Reporting and Recordkeeping Requirements and Test Rule; 52 FR 

21018, June 4, 1987) and was found to have the following deficiencies compared with the EPA 

797.1400 test guideline: (1) the age of the fish was not reported; (2) the acidity, total organic 

carbon or chemical oxygen demand, and particulate matter of the dilution water were not 

reported; (3) the report did not indicate which pesticides were measured analytically or provide 

data on other possible contaminants in the dilution water; (4) the test temperature varied from the 

guideline-specifed range of 12 ± 2°C for cold-water species (range during test: 13-15°C); (5) 18 

measurements of dissolved oxygen concentrations were less than 8.2 mg/L, whereas the 

guideline specifies dissolved oxygen shall be maintained above 8.2 mg/L in flow-through tests 

with cold-water species such as trout; (6) at 0 hours, there was a relatively large difference in pH

levels (up to 0.6 pH units) between replicates for most groups, including the controls; (7) the 

measured test concentrations at 96 hours in replicates A and B at nominal concentrations of 100, 

27 and 18 μg/L varied by more than the maximum of 20% allowed by the guideline; (8) between 

 

1 Percent decrease was not calculated for one replicate in which the measured concentration at 96 hours was below 

the detection limit.
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0 hours and 96 hours, the measured test concentration at the nominal concentration of 18 μg/L

varied by more than the maximum of 30% allowed by the guideline; (9) the concentration of 

acetone used (75 μL/L) exceeded the test guideline maximum of 5 mg/L (equivalent to ~ 6.2 μL

acetone/L); (10) the water solubility determinations and adequacy of the methods to attain 

maximum test concentrations were not adequately explained; and (11) the maximum water 

solubility at the highest test concentration was not achieved based on a validated water solubility 

study conducted after the toxicity test, i.e., the highest test concentration of 45 μg/L was ~59% of

the measured maximum water solubility of 76.1 μg/L at a temperature and pH closely 

approximating the fish test conditions (12 °C and pH of 7.0). 

REFERENCES

Springborn Life Sciences, Inc. 1988b. Acute Toxicity of Anthraquinone to Coho Salmon 

(Orcorhynchus kisutch) under Flow-Through Conditions. Report # 88-9-2812. SLS Study 

#274.0488.6176.157. Sponsor: Mobay Corporation. 

Text in quotes is taken directly from Springborn Life Sciences, Inc., 1988b.
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Study 5: Acute toxicity study in bluegill sunfish (Springborn Life Sciences, Inc., 1988c) 

Title: Acute Toxicity of Anthraquinone to Bluegill Sunfish (Lepomis macrochirus) Under FlowThrough Conditions

This study is: Adequate Not Adequate

The study had the following deficiencies compared with the EPA 797.1400 test guideline: (1) the 

age of the fish was not reported; (2) the acidity, total organic carbon or chemical oxygen 

demand, and particulate matter of the dilution water were not reported; (3) the report did not 

indicate which pesticides were measured analytically or provide data on other possible 

contaminants in the dilution water; (4) dissolved oxygen levels were ≤ 6.6 mg/L in 39 out of 210 

measurements, whereas the guideline specifies dissolved oxygen shall be maintained above 6.6 

mg/L in flow-through tests with bluegill; (5) the concentration of acetone used (75 μL/L) 

exceeded the test guideline maximum of 5 mg/L (equivalent to ~ 6.2 μL acetone/L); (6) the test 

concentration was not measured on Day 10 when the diluter malfunctioned for 21 hours; (7) the 

measured test concentrations at 0 hours in replicates A and B at the nominal concentration of 18 

μg/L varied by more than the maximum of 20 percent allowed by the guideline; (8) the water 

solubility determinations and adequacy of the methods to attain maximum test concentrations 

were not adequately explained; (9) the maximum water solubility at the highest test 

concentration was not achieved based on a validated water solubility study conducted after the 

toxicity test, i.e., the highest test concentration of 45 μg/L was approximately 36% of the 

measured water solubility of 125 μg/L at a temperature and pH closely approximating the fish 

test conditions (22 °C and pH of 7.2); and (10) the fish were not fed between days 5 and 9, 

whereas the guideline specifies feeding every other day beginning on day 5.

TEST SUBSTANCE 

Identity: 9,10-Anthracenedione, CASRN 84-65-1 

Remarks: Purity: 99.81% active ingredient (A.I.) according to the study sponsor; 50:50 

mixture of Mobay and ICI anthraquinone powders (Mobay lot #2025865; ICI lot# 611884);

yellow powder received by the Mobay Corporation.

METHOD

Method/guideline followed: TSCA Guidelines (number of guideline not specified)

Test type: Prolonged toxicity to fish (flow-through)

GLP compliant?: Yes

Year study was performed: 1988

Test species: Bluegill sunfish (Lepomis macrochirus)
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Analytical monitoring: Yes; HPLC

Exposure duration: 15 days 

Concentrations tested: Nominal concentrations: 0, 18, 27, 42, 65, and 100 μg a.i./L; Mean 

measured concentrations: 0, 12, 16, 23, 34, and 45 μg a.i./L.

Test method/conditions remarks: “The test was conducted using an exposure system 

consisting of a constant flow serial diluter..., a temperature-controlled water bath, and a set of 14 

exposure aquaria... [Twenty organisms were exposed in duplicate test aquaria (10 fish per 

replicate) in a flow-through system] to five concentrations of the test substance, a dilution water 

control, and a solvent control. The solvent control solutions contained the maximum amount of 

acetone... present in any test concentration (75 μL/L). All treatment levels and the controls were 

maintained in duplicate. Test aquaria were labelled to identify the test sample, nominal test

concentration and designated replicate. Test solutions were not aerated. A photoperiod of 16 

hours light and 8 hours dark with a light intensity of 32-60 footcandles at the test solution surface 

was maintained throughout the test period. Lighting was provided by Durotest Vita-LiteR

fluorescent bulbs. [Springborn Life Sciences, Inc. 1988c, p. 8-9]

“Concentrations for this study were based on the limit of water solubility of the test substance in 

freshwater, under the maintained test conditions. Prior to initiation of the definitive study, 

preliminary testing was conducted at Springborn Life Sciences, Inc. (SLS) to establish the water 

solubility and acute toxicity of anthraquinone under test conditions. Test conditions for this 

study were generally consistent with the requirements published in the EPA ́s Final Test Rule, 

Parts 704, 795, and 799 (Federal Register Volume 52, No. 107, 4 June 1987). Preliminary 

testing corroborated with information provided by the Mobay Corporation established that the 

limit of water solubility for the test substance, under the maintained test conditions, was 

approximately 50 μg a.i./L.... Based on these data, the following nominal concentrations were 

selected for the 14-day definitive study: 18, 27, 42, 65, and 100 μg a.i./L.” [Springborn Life 

Sciences, Inc. 1988b, p. 4] 

“Each glass test aquarium measured 39 x 20 x 25 centimeters (cm) with a 19.5 cm high 

standpipe, which maintained a constant test water volume of 15 L. The diluter was constructed 

to deliver 68 mL of solution per minute to each replicate test aquarium, providing approximately 

6.5 volume replacements per aquarium every 24 hours. Test aquaria were impartially positioned 

in a water bath containing circulating water designed to maintain the test solution temperatures at 

22 ± 2°C.” [Springborn Life Sciences, Inc. 1988c, p. 10] 

“The test was initiated when ten bluegill were impartially selected and distributed to each 

replicate aquarium. The maximum organism loading concentration, (throughout the test) was 

0.17 g of biomass per liter of flowing test solution per day.” [Springborn Life Sciences, Inc. 

1988c, p. 10]

Biological observations were made and recorded at test initiation and every 24 hours thereafter 

until the test was terminated.
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During the first 14 days of the study, the pH ranged from 6.9 to 7.7, dissolved oxygen ranged 

from 2.7 to 8.7 mg/L, and the temperature ranged from 21 to 23°C. Water quality measurements 

were not provided for the 15th day of exposure. 

Details on Sampling: “During the in life phase of the definitive study, water samples were 

removed from both replicate test solutions of each treatment level and the controls on test days 0 

and 4 for analysis of Anthraquinone. Subsequently, on days 7 and 14, water samples were 

removed from both replicate test solutions of the two highest test concentrations and the control 

solutions. Each exposure solution sample was collected from the approximate midpoint of the 

aquarium with a volumetric pipet. In addition, quality assurance (QA) blind samples were 

prepared at each sampling interval and remained with the set of exposure solution samples 

through the analytical process. The QA samples were prepared in dilution water at 

Anthraquinone concentrations unknown to the analyst. Results of these analyses were used to 

judge the precision and quality control maintained during the analysis of exposure solution 

samples.” [Springborn Life Sciences, Inc. 1988c, p. 12]

Details on Test Solutions: “A diluter stock solution of 1.34 mg A.I./mL was prepared by diluting 

0.3358 g of Anthraquinone (0.3352 grams active ingredient [A.I.]) with acetone to a total volume 

of 250 mL. A Sage syringe pump in conjunction with a 50-mL gas-tight syringe was used to 

deliver 0.029 mL/min of the stock solution of Anthraquinone (1.34 mg A.I./mL) to the diluter's 

chemical mixing chamber receiving approximately 0.389 L/min dilution water. The mixing 

chamber was positioned over a magnetic stirrer, with an ultrasonic water bath. The continuous 

stirring and sonication aided in the solubilization of the test material. The solution contained in 

the mixing chamber constituted the highest nominal test concentration (100 μg A.I./L) and was 

subsequently diluted (65% dilution factor) to provide the desired exposure concentration range. 

A similar system used to deliver the test material was also used to deliver acetone to the solvent 

control aquaria.” [Springborn Life Sciences, Inc. 1988c, p. 9-10]

Details on Test Organisms: “The bluegill (SLS lot #88A9A) were obtained from a commercial 

supplier in Missouri. Prior to testing, these fish were held in a 500-L fiberglass tank under a 

photoperiod of 16 hours light and 8 hours darkness. The well water that flowed into this holding 

tank was characterized as having a total hardness and alkalinity range as calcium carbonate 

(CaCO3) of 27-32 mg/L and 22-24 mg/L, respectively, and a specific conductance range of 100-

130 micromhos per centimeter (μmhos/cm) (Weekly Gravity Feed Tank Water Quality Analysis 

Log Book).. Other parameters monitored in the holding tank included pH with a range of 6.7-

7.1, dissolved oxygen concentrations... of 75-80% saturation, and a flow rate of 6.9-7.5 tank 

volume replacements/day (Weekly Record of Fish Holding Characteristics). Test fish were 

maintained under similar conditions for a minimum of 14 days prior to testing. The temperature 

range in the holding tank was 21-23°C during this 14-day period. All fish were fed a 

commercial pelleted food, ad libitum, daily except during the 48 hours prior to testing. Fish 

were also fed on days 5, 9, 11, and 13 of the exposure. There was ≤ 0.1% mortality in the test 

fish population during the 2 days prior to testing (Daily Record of Fish Holding Conditional.

The bluegill used during this study were all of the same year class and a representative sample 

(n=30) of the fish from this population had a mean (range) wet weight of 1.7 (1.09-2.45) grams 

and total length of 50 (42-59) millimeters (Fish Weight and Lengths Log).” [Springborn Life 

Sciences, Inc. 1988c, p. 7-8]
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Protocol deviations were reported and included low dissolved oxygen (dropped below 6.6 mg/L; 

range during test was 2.7-8.7 mg/L), incorrect feeding schedule (should have occurred every 

other day but no feeding occurred between days 5 and 9), use of a flow serial diluter instead of a 

proportional diluter, and occurrence of diluter malfunction on test day 10, which resulted in no 

test material being delivered for approximately 21 hours (and concentrations of test substance in 

test solutions were not analytically confirmed until day 14). 

RESULTS

Endpoint values: 15-day LC50 > 45 μg a.i./L (based on mean measured concentrations); 15-day 

NOEC = 45 μg a.i./L (based on mean measured concentrations)

Results remarks: “Throughout the exposure period, a small amount of precipitate was observed 

in the diluter system's mixing chamber. However, no undissolved anthraquinone (e.g., 

precipitate, film on solution's surface) was observed in any of the exposure vessels during the 

study.” [Springborn Life Sciences, Inc. 1988c, p. 15-16]

Measured test concentrations at the nominal concentration of 18 μg/L varied more than 20% 

between replicates A and B at 0 hours; the difference between replicates was 27%.

During 15 days of exposure, two fish died at 34 μg a.i./L and one fish died in the solvent control. 

No mortalities were observed at other test concentrations. Sublethal effects included darkened 

pigmentation (in 2 fish at 34 μg a.i./L and 1 fish at 12 μg a.i./L), partial loss of equilibrium (in 1 

fish at 34 μg a.i./L), and presence at the surface of the test solution (in 2 fish at 34 μg a.i./L). 

The study authors did not consider the mortalities and sublethal effects to represent adverse 

effects of the test substance.

Due to the diluter malfunction on day 10, the exposure was extended by 24 hours to a total of 15 

days. No additional mortalities or sublethal effects were observed during the final 24 hours of 

exposure.

CONCLUSIONS

“Based on this [sic] data, it was established that under the test conditions maintained during this 

study (e.g., water quality, solution temperature, solvent concentration, mixing period), the test 

substance was not acutely toxic to bluegill at and below the material's limit of water solubility. 

The 15-day LC50 for the test substance in bluegill was empirically estimated as being greater 

than the highest mean measured test concentration (45 μgA.I./L).” [Springborn Life Sciences, 

Inc. 1988c, p. 17]

STUDY RELIABILITY

[3] Not reliable. This study was submitted in response to EPA’s 1987 test rule for CASRN 84-

65-1 (Anthraquinone; Final Reporting and Recordkeeping Requirements and Test Rule; 52 FR 

21018, June 4, 1987) and was found to have the following deficiencies compared with the EPA 
Draft HPV1/HPV2 Chemical Data Adequacy Review (May 2013) CASRN 84-65-1

22

797.1400 test guideline: (1) the age of the fish was not reported; (2) the acidity, total organic 

carbon or chemical oxygen demand, and particulate matter of the dilution water were not 

reported; (3) the report did not indicate which pesticides were measured analytically or provide 

data on other possible contaminants in the dilution water; (4) dissolved oxygen levels were ≤ 6.6 

mg/L in 39 out of 210 measurements, whereas the guideline specifies dissolved oxygen shall be 

maintained above 6.6 mg/L in flow-through tests with bluegill; (5) the concentration of acetone 

used (75 μL/L) exceeded the test guideline maximum of 5 mg/L (equivalent to ~ 6.2 μL 

acetone/L); (6) the test concentration was not measured on Day 10 when the diluter 

malfunctioned for 21 hours; (7) the measured test concentrations at 0 hours in replicates A and B 

at the nominal concentration of 18 μg/L varied by more than the maximum of 20 percent allowed 

by the guideline; (8) the water solubility determinations and adequacy of the methods to attain 

maximum test concentrations were not adequately explained; (9) the maximum water solubility 

at the highest test concentration was not achieved based on a validated water solubility study 

conducted after the toxicity test, i.e., the highest test concentration of 45 μg/L was approximately 

36% of the measured water solubility of 125 μg/L at a temperature and pH closely 

approximating the fish test conditions (22 °C and pH of 7.2); and (10) the fish were not fed 

between days 5 and 9, whereas the guideline specifies feeding every other day beginning on day 

5. 

REFERENCES

Springborn Life Sciences, Inc. 1988c. Acute Toxicity of Anthraquinone to Bluegill Sunfish 

(Lepomis macrochirus) Under Flow-Through Conditions. Report # 88-11-2855. SLS Study 

#274.0488.6176.105. Sponsor: Mobay Corporation. 

Text in quotes is taken directly from Springborn Life Sciences, Inc., 1988c.
Draft HPV1/HPV2 Chemical Data Adequacy Review (May 2013) CASRN 84-65-1

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Study 6: Acute toxicity study in fathead minnow (Geiger et al., 1988)

Title: Acute toxicities of organic chemicals to fathead minnows (Pimephales promelas)

This study is: Adequate Not Adequate

This study is not adequate because group size was 5 fish rather than the 10 fish recommended by 

ASTM E729-96 or 7 fish recommended by OECD TG 203 and OPPTS 850.1075. Also, 

analytical measurements of test substance were not conducted at 0 and 24 hours.

TEST SUBSTANCE 

Identity: 9,10-Anthracenedione, CASRN 84-65-1

Remarks: 97% pure; source: Aldrich Chemical Co.

METHOD

Method/guideline followed: Not Specified

Test type: Acute toxicity to fish (flow-through)

GLP compliant?: Not Specified

Year study was performed: 1985

Test species: Fathead minnow (Pimephales promelas), mean length: 18.5 ± 1.4 mm; mean 

weight: 0.086 ± 0.021 g; age: 29 days old

Analytical monitoring: Yes; HPLC

Exposure duration: 96 Hours

Concentrations tested: 0.24 mg/L (mean measured)

Test method/conditions remarks: In a limit test, groups of five fathead minnows were exposed 

to 0 (control) or 0.24 mg 9,10-anthracenedione /L in a total volume of 250 mL (freshwater 

media) for 96 hours. A modified continuously proportioning diluter was used in this flowthrough test. The flow rate was 28.8 volumes/day. The loading rate was 1.72 g fish/L. 

Measured concentrations of test substance were 0.243, 0.234, and 0.254 mg/L at 48, 72, and 96 

hours, respectively. The mean measured concentration was 0.24 mg/L. According to the study 

authors, the solution was 96% saturated. Previously reported water solubility limit values are 
Draft HPV1/HPV2 Chemical Data Adequacy Review (May 2013) CASRN 84-65-1

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74.6 μg/L (20°C, pH 7; Case Consulting Laboratories, 20061) and 1.35 mg/L (25 °C; Yalkowsky

and Dannenfelser, 19902). 

Alkalinity was 46.0 CaCO3/L, water hardness was 43.0 mg CaCO3/L, dissolved oxygen was 7.1 

± 0.12 mg/L, pH was 7.7, and the temperature was 24.0 ± 0.015°C. 

RESULTS

Endpoint value(s): 96-hour LC100 > 0.24 mg/L (based on measured concentration)

Results remarks: There was no mortality in either the control or the one test concentration of 

0.24 mg/L. Exposed fish showed no evidence of stress during the test.

CONCLUSIONS

The 96-hour LC100 for fathead minnow was > 0.24 mg/L.

STUDY RELIABILITY

[3] Not reliable. Group size was 5 fish rather than the 10 fish recommended by ASTM E729-96. 

Also, analytical measurements of test substance were not conducted at 0 and 24 hours.

REFERENCES

Geiger, D.L., Call, D.J., and Brooke, L.T. 1988. Acute Toxicities of Organic Chemicals to 

Fathead Minnows (Pimephales promelas), Vol. IV. Center for Lake Superior Environmental 

Studies, University of Wisconsin-Superior. Available at 

http://www.epa.gov/pesticides/chem_search/reg_actions/registration/fs_PC-122701_01-Dec98.pdf as of May 22, 2013.

 

1 Case Consulting Laboratories, Inc. 2006. Physical and Chemical Characteristics of Anthraquinone: 

Octanol/Water Partition Coefficient and Solubility. Sponsor: Chemical Products Technologies, LLC. Study No. 

3320-02. November 20, 2006. 2 Yalkowsky, S.H and Dannenfelser, R.M. 1990. AQUASOL Database of Aqueous Solubility. Fifth edition. 

Tucson, AZ: University of Arizona, College of Pharmacy. Current book version: Yalkowsky, F.H. and He, Y. 

2003. Handbook of Aqueous Solubility Data. CRC Press, Boca Raton, FL (ISBN 0-8493-1532-8). Value obtained 

from EPI Suite v4.1.
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Study 7: Acute toxicity study in northern squawfish, Chinook salmon, and coho salmon

(MacPhee and Ruelle, 1969) 

Title: Lethal effects of 1888 chemicals upon four species of fish from western North America

This study is: Adequate Not Adequate

This study is not adequate because: Analytical monitoring was not performed and only one fish

per species was exposed to the test substance. There was no evidence that a control was used. 

An LC50 was not determined, only an LC100.

TEST SUBSTANCE 

Identity: 9,10-Anthracenedione, CASRN 84-65-1

Remarks: Purity not provided

METHOD

Method/guideline followed: No data

Test type: Acute static fish test

GLP compliant?: Pre-GLP

Year study was performed: 1969

Test species: Northern squawfish (Ptychocheilus oregonensis), Chinook salmon (Oncorhynchus 

tshawytscha), and coho salmon (Oncorhynchus kisutch). Fish ranged in size from 5 to 10 cm in 

length.

Analytical monitoring: No

Exposure duration: 24 hours

Concentrations tested: 10 mg/L

Test method/conditions remarks: The dilution water utilized for this test was collected from 

Rochat Creek. One squawfish and one of each of the two species of salmonid were exposed in

9.5-L plastic aquaria containing 4 L of test solution for 24 hours during a limit test. The loading 

rate was ~ 5 g fish/L. The times at which the fish lost their equilibrium and died were recorded. 

The test was conducted at a concentration above the reported water solubility limit values of 74.6 
Draft HPV1/HPV2 Chemical Data Adequacy Review (May 2013) CASRN 84-65-1

26

μg/L (20°C, pH 7; Case Consulting Laboratories, 20061) and 1.35 mg/L (25 °C; Yalkowsky and 

Dannenfelser, 19902). 

Acetone was used as a solvent for “most” of the chemicals tested in this study, but the authors do 

not explicitly state whether acetone was used as a solvent in the test of CASRN 84-65-1.

Alkalinity was 7 ppm, water hardness ranged from 0 to 17 ppm, pH was 7.2, and temperature 

was 52 °F. 

RESULTS

Endpoint value(s): 24-hour LC100 ≤ 10 mg/L (nominal concentration)

Results remarks: The squawfish lost equilibrium between 5 and 9 hours after test initiation, 

and died within 9-13 hours after test initiation. The Chinook salmon and coho salmon both died 

between 5 and 9 hours after test initiation.

CONCLUSIONS

The 24-hour LC100 for Ptychocheilus oregonensis, Oncorhynchus tshawytscha, and 

Oncorhynchus kisutch was ≤ 10 mg/L.

STUDY RELIABILITY

[3] Not reliable. Analytical monitoring was not performed and only one fish per species was 

exposed to the test substance. There was no evidence that a control was used. This was a limit 

test. Study duration was meant to be 24 hours but all fish died between 5 and 13 hours after test 

initiation. An LC50 was not determined, only an LC100. Data are useful for performing a future 

definitive test. 

REFERENCES

MacPhee, C. and Ruelle, R. 1969. Lethal Effects of 1888 Chemicals upon Four Species of Fish 

from Western North America. Bulletin No. 3. University of Idaho College of Forestry -

Wildlife and Range Sciences.

 

1 Case Consulting Laboratories, Inc. 2006. Physical and Chemical Characteristics of Anthraquinone: 

Octanol/Water Partition Coefficient and Solubility. Sponsor: Chemical Products Technologies, LLC. Study No. 

3320-02. November 20, 2006. 2 Yalkowsky, S.H and Dannenfelser, R.M. 1990. AQUASOL Database of Aqueous Solubility. Fifth edition. 

Tucson, AZ: University of Arizona, College of Pharmacy. Current book version: Yalkowsky, F.H. and He, Y. 

2003. Handbook of Aqueous Solubility Data. CRC Press, Boca Raton, FL (ISBN 0-8493-1532-8). Value obtained 

from EPI Suite v4.1.
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Study 8: Acute toxicity study in Mozambique tilapia (Jaiswal et al., 1987)

Title: Annotation of comparative median lethal concentrations of few oxygen heterocyclic 

compounds for Oreochromis mossambicus (Peters) Trewavas and changes induced by these 

biocides

This study is: Adequate Not Adequate

This study is not adequate because: Mortality was not reported; it is not clear how many fish 

died during exposure. Toxic endpoint values were not derived, but were taken from another 

article. Analytical monitoring was not performed. An LC50 was not determined. The exposure 

duration was only 48 hours, rather than 96 hours.

TEST SUBSTANCE 

Identity: 9,10-Anthracenedione, CASRN 84-65-1

Remarks: Purity not provided

METHOD

Method/guideline followed: No data

Test type: Acute static fish test, open test vessel

GLP compliant?: No

Year study was performed: 1987

Test species: Mozambique tilapia (Oreochromis mossambicus)

Analytical monitoring: No

Exposure duration: 48 hours

Concentrations tested: 13.5 mg/L

Test method/conditions remarks: Ten fish were exposed to 13.5 mg/L (nominal) of the test 

substance. This 48-hour median lethal concentration was determined by Busvine in 1975. The 

test temperature was between 26 and 32°C. The test was conducted at a concentration above the 
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reported water solubility limit values of 74.6 μg/L (20°C, pH 7; Case Consulting Laboratories, 

20061) and 1.35 mg/L (25 °C; Yalkowsky and Dannenfelser, 19902).

 

RESULTS

Endpoint value(s): 48-hour LC100 > 13.5 mg/L (value determined by Busvine in 1975)

Results remarks: At time of death, the fish were bled and were shown to have a significantly 

higher red blood cell count compared to the controls. It is not stated how long the fish were 

exposed to 13.5 mg/L of the test substance before they died.

CONCLUSIONS

The 48-hour LC100 was > 13.5 mg/L (value determined by Busvine in 1975).

STUDY RELIABILITY

[3] Not reliable. Toxic endpoint values were not derived, but were taken from another article. 

Mortality was not reported; it is not clear how many fish died during exposure. It was not 

reported how long the fish were exposed to 13.5 mg/L of the test chemical before they died.

Analytical monitoring was not performed. An LC50 was not determined. The exposure duration 

was only 48 hours, rather than 96 hours. 

REFERENCES

Jaiswal, S.K. and Waghray, S. 1989. Annotation on Comparative Median Lethal Concentrations 

of Few Oxygen Heterocyclic Compounds for Oreochromis mossambicus (Peters) Trewavas and 

Changes Induced by these Biocides. J. Environ. Biol. 10: 393-398. As summarized in HPVIS.

 

1 Case Consulting Laboratories, Inc. 2006. Physical and Chemical Characteristics of Anthraquinone: 

Octanol/Water Partition Coefficient and Solubility. Sponsor: Chemical Products Technologies, LLC. Study No. 

3320-02. November 20, 2006. 2 Yalkowsky, S.H and Dannenfelser, R.M. 1990. AQUASOL Database of Aqueous Solubility. Fifth edition. 

Tucson, AZ: University of Arizona, College of Pharmacy. Current book version: Yalkowsky, F.H. and He, Y. 

2003. Handbook of Aqueous Solubility Data. CRC Press, Boca Raton, FL (ISBN 0-8493-1532-8). Value obtained 

from EPI Suite v4.1.
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C-2. Acute Toxicity to Aquatic Invertebrates

Summary of endpoint: Adequate

Study 1: Acute toxicity study in Daphnia (EPA, 2006)

Title: Anthraquinone (122701) Fact Sheet, Biopesticides and Pollution Prevention Division 

Office of Pesticide Programs

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: 9,10-Anthracenedione, CASRN 84-65-1

Remarks: Purity not provided; test substance identified as anthraquinone

METHOD

Method/guideline followed: No data

Test type: No data

GLP compliant?: No data

Year study was performed: No data

Test species: Water flea (Daphnia species)

Analytical monitoring: No data

Exposure duration: 48 hours 

Concentrations tested: No data

Test method/conditions remarks: No data

RESULTS

Endpoint value(s): 48-hour LC50 > 240 μg/L; 48-hour NOEC > 240 μg/L

 

Results remarks: The 48-hour LC50 for Daphnia is > 240 μg/L and the NOEC is > 240 μg/L; 

all daphnids survived the study period and no sublethal effects were observed.

The Office of Pesticide Programs “Anthraquinone (122701) Fact Sheet” reports the LC50 and 

NOEC in units of “Fg/L”, i.e., LC50 > 240 Fg/L and NOEC > 240 Fg/L. The meaning of “Fg” is 
Draft HPV1/HPV2 Chemical Data Adequacy Review (May 2013) CASRN 84-65-1

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not defined in the Fact Sheet. Based on the solubility limit of CASRN 84-65-1, the units are 

assumed to be μg/L; the use of “Fg/L” rather than μg/L appears to be a transcription error. The 

original study and the Data Evaluation Review for this study were not available for review; the 

study report is considered to be FIFRA proprietary information.

CONCLUSIONS

The 48-hour LC50 for Daphnia was > 240 μg/L.

STUDY RELIABILITY

[1] Reliable without restrictions. This study, MRID # 44496124, is considered acceptable by 

OPP.

REFERENCES

EPA. 2006. Anthraquinone (122701) Fact Sheet, Biopesticides and Pollution Prevention 

Division Office of Pesticide Programs, EPA, Washington, DC. Available at 

http://www.epa.gov/pesticides/chem_search/reg_actions/registration/fs_PC-122701_01-Dec98.pdf as of May 22, 2013.
Draft HPV1/HPV2 Chemical Data Adequacy Review (May 2013) CASRN 84-65-1

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Study 2: Acute toxicity study in Daphnia magna (Springborn Life Sciences, Inc. 1988d)

Title: Acute Toxicity of Anthraquinone to Daphnids (Daphnia magna) Under Flow-through 

Conditions

This study is: Adequate Not Adequate

The study had the following deficiencies compared with EPA 797.1300 test guideline: (1) there 

is no indication as to whether the test chambers were covered as stipulated in the guideline; (2) 

the total organic carbon or chemical oxygen demand, and particulate matter of the dilution water 

were not reported; (3) the report did not indicate which pesticides were measured analytically or 

provide data on other possible contaminants in the dilution water; (4) the report did not indicate 

whether or not all test equipment was cleaned; (5) the water solubility determinations and 

adequacy of the methods to attain maximum test concentrations were not adequately explained; 

(6) the maximum water solubility at the highest test concentration was not achieved based on a 

validated water solubility study conducted after the toxicity test, i.e.,the highest test 

concentration of 48 μg/L was approximately 38% of the measured maximum water solubility of 

125 μg/L at a temperature and pH closely approximating the daphnid test conditions (22 °C and 

pH of 7.2); and (7) the concentration of acetone used (91 μL/L) exceeded the test guideline 

maximum of 0.1 mg/L (equivalent to ~ 0.12 μL acetone/L).

TEST SUBSTANCE 

Identity: 9,10-Anthracenedione, CASRN 84-65-1 

Remarks: Purity: 99.81% active ingredient (A.I.) according to the study sponsor; 50:50 

mixture of Mobay and ICI anthraquinone powders (Mobay lot #2025865; ICI lot# 611884);

yellow powder received by the Mobay Corporation.

METHOD

Method/guideline followed: TSCA Guidelines (number of guideline not specified)

Test type: Daphnia sp., Acute Immobilization Test (flow-through)

GLP compliant?: Yes

Year study was performed: 1988 

Test species: Water flea (Daphnia magna), ≤ 24 hours old

Analytical monitoring: Yes; HPLC

Exposure duration: 48 hours

Concentrations tested: Nominal concentrations: 0, 13, 22, 36, 60, and 100 μg a.i./L;

Mean measured concentrations: 0, 6.9, 10, 18, 27, and 48 μg a.i./L. 
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Test method/conditions remarks: “The test was conducted using an exposure system 

consisting of an intermittent-flow proportional diluter... and a set of 14 exposure vessels. The 

test system was designed to provide five concentrations of the test material, a dilution water 

control, and a solvent control. The solvent control solution contained the maximum amount of 

acetone... present in any test concentration (91 μL acetone/L). Exposure vessels were 

maintained in an area illuminated with cool White and “GrO-Lux” fluorescent light at an 

intensity of 30-50 footcandles and a photoperiod of 16 hours light and 8 hours dark... Two 

replicate vessels were established for each treatment level and the controls. Test aquaria were 

labeled to identify the test sample nominal concentration and designated replicate.” [Springborn 

Life Sciences, Inc. 1988d, p. 8-9]

“The test was initiated when ten daphnids (≤24 hours old), twenty per treatment level and 

controls, were impartially selected and introduced to each exposure vessel.” [Springborn Life 

Sciences, Inc. 1988d, p. 11]

Two control groups (dilution water control and solvent control) were tested concurrently. The 

number of immobilized daphnids observed in each replicate test solution was recorded at 24 and 

48 hours during the exposure. Water quality measurements made daily during the 48-hour 

definitive test established that the parameters measured (pH, dissolved oxygen, temperature, 

specific conductance, hardness and alkalinity) were consistent throughout the exposure and were 

unaffected by the concentrations of the test substance tested. Continuous monitoring in one 

replicate of the 100 μg a.i./L (nominal) treatment level established that the temperature was 19-

22°C during the exposure period.

“Prior to initiation of the definitive study, preliminary testing was conducted at Springborn Life 

Sciences, Inc. (SLS) to establish the water solubility and acute toxicity of the test substance 

under test conditions. Test conditions for this study were consistent with the requirements 

published in the EPA's Final Test Rule, Parts 704, 795, and 799 (Federal Register Volume 52, 

No. 107, 4 June 1987). Preliminary testing corroborated information provided by the Mobay 

Corporation and established that the limit of water solubility for the test substance, under the 

maintained test conditions, was approximately 50 μg A.I./L. During this same preliminary test 

period, it was also determined that concentrations of ≤ 50 μg A.I./L test substance were not 

acutely toxic to daphnids. Based on these data, the following nominal concentrations were 

selected for the 48-hour definitive study: 13, 22, 36, 60, and 100 μg A.I./L.” [Springborn Life 

Sciences, Inc. 1988d, p. 14]

The total hardness was 170-180 mg CaCO3/L, the test temperature was 19-20°C, the pH was 

8.1-8.4, and the dissolved oxygen concentration was 7.7-9.1 mg/L.

A small amount of precipitate was observed in the diluter system's mixing chamber and was 

removed as necessary. No visible sign of undissolved material (e.g., precipitate, film on solution 

surface) was observed in any of the exposure vessels. Biological observations were made and 

recorded at test initiation and every 24 hours thereafter until the test was terminated.
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Details on Test Solutions: Concentrations of the test material were maintained in the exposure 

vessels by introducing approximately 6 aquarium volumes per day of newly prepared test 

solution via an intermittent-flow proportional diluter. “A diluter stock solution of 1.10 mg A.I./L 

was prepared by diluting 0.1102 g Anthraquinone (0.1100 grams active ingredient [A.I.]) with 

acetone to a total volume of 100 mL. The diluter was equipped with a 50-mL gas-tight GlancoR

syringe which delivered 0.0420 mL of the stock solution (1.10 mg A.I./mL) into 463 mL of 

dilution water in the exposure system's mixing chamber during each diluter cycle. This 463-mL 

solution served at the highest treatment level (100 μg A.I./L, nominal) from which calibrated 

volumes were diluted to provide the 60% nominal concentration gradient (100-13 μg A.I./L of 

test substance).” [Springborn Life Sciences, Inc. 1988d, p. 9-10] 

Details on Sampling: “Water samples were removed from both replicate vessels of the control 

and the high, middle, and low test concentrations once prior to initiating the definitive exposure. 

Results of these analyses were used to judge whether sufficient quantities of the test substance 

were being delivered to the test vessels and appropriate test concentrations were being 

maintained to initiate the definitive exposure. During the in-life phase of the test, water samples 

were removed and analyzed for the test substance on day 0 and the day of the test termination 

(day 2) from both replicate vessels of each test concentration and the controls.” [Springborn Life 

Sciences, Inc. 1988d, p. 12-13]

Three quality assurance (QA) samples, formulated in test dilution water to contain 

concentrations of the test substance unknown to the analyst, were also prepared at each sampling 

interval.

Details on Test Organisms: “The daphnids used in this toxicity test were obtained from 

laboratory cultures maintained at Springborn Life Sciences, Inc., Wareham, Massachusetts. The 

culture water was prepared by fortifying well water based on the formula for hard water (ASTM 

1980) and filtering it through an Amberlite XAD-7 resin column to remove any potential organic 

contaminants. This water generally had total hardness and alkalinity ranges as calcium carbonate 

(CaCO3) of 160-180 mg/L and 110-130 mg/L, respectively, a pH range of 7.9-8.3, a temperature 

of 20 ± 1°C, a dissolved oxygen concentration of greater than 60% of saturation, and a specific 

conductivity range of 400-600 micromhos per cm (μmhos/cm).” [Springborn Life Sciences, Inc.

1988d, p.7]

Deviations from Protocol: “The protocol states that the calibration of the diluter system is 

checked prior to test initiation and at test termination. During this study, the calibration of the 

diluter was not checked at test termination; however, stock consumption and analytical 

measurements of test concentrations verify that the diluter was functioning properly throughout 

the 48-hour study.” [Springborn Life Sciences, Inc. 1988d, p. 18] The study author believed that 

this deviation did not affect the results of this study.

RESULTS

Endpoint value(s): 48-hour EC50 > 48 μg a.i./L (based on mean measured concentrations); 

48-hour NOEC = 48 μg a.i./L (based on mean measured concentrations). 
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Results remarks: Following 48-hours of exposure, no immobilization and no adverse effects 

were observed among daphnids exposed to the test substance (6.9-48 μg a.i./L). Ten percent 

immobilization was observed in both the control and solvent control groups. At 24 hours, 

entrapment of several daphnids at the surface of the test solution was observed in the solvent 

control.

CONCLUSIONS

“Based on this [sic] data, it was established that under the test conditions maintained during this 

study (e.g., water quality, solution temperature, solvent concentration, mixing period), 

Anthraquinone was not acutely toxic to daphnids at and below the material's limit of water 

solubility. The 48-hour EC50 for the test substance and daphnids was empirically estimated at 

being greater than the highest mean measured test concentration (48 μg A.I./L). The NoObserved-Effect Concentration established for this study was 48 μg A.I./L, the highest mean 

measured concentration tested.” [Springborn Life Sciences, Inc. 1988d, p. 17] 

STUDY RELIABILITY

[3] Not reliable. This study was submitted in response to EPA’s 1987 test rule for CASRN 84-

65-1 (Anthraquinone; Final Reporting and Recordkeeping Requirements and Test Rule; 52 FR 

21018, June 4, 1987) and was found to have the following deficiencies compared with EPA 

797.1300 test guideline (with modifications indicated in EPA’s 1987 test rule for anthraquinone: 

Anthraquinone; Final Reporting and Recordkeeping Requirements and Test Rule; 52 FR 21018):

(1) there is no indication as to whether the test chambers were covered as stipulated in the 

guideline; (2) the total organic carbon or chemical oxygen demand, and particulate matter of the 

dilution water were not reported; (3) the report did not indicate which pesticides were measured 

analytically or provide data on other possible contaminants in the dilution water; (4) the report 

did not indicate whether or not all test equipment was cleaned; (5) the water solubility 

determinations and adequacy of the methods to attain maximum test concentrations were not 

adequately explained; and (6) the maximum water solubility at the highest test concentration was 

not achieved based on a validated water solubility study conducted after the toxicity test, i.e.,the 

highest test concentration of 48 μg/L was approximately 38% of the measured maximum water 

solubility of 125 μg/L at a temperature and pH closely approximating the daphnid test conditions 

(22 °C and pH of 7.2). 

REFERENCES

Springborn Life Sciences, Inc. 1988d. Acute Toxicity of Anthraquinone to Daphnids (Daphnia 

magna) Under Flow-Through Conditions. Report # 88-12-2885. SLS Study #274.0488.6176.115. 

Sponsor: Mobay Corporation. 

Text in quotes is taken directly from Springborn Life Sciences, Inc., 1988d.
Draft HPV1/HPV2 Chemical Data Adequacy Review (May 2013) CASRN 84-65-1

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Study 3: Acute toxicity study in Crassostrea virginica (Springborn Life Sciences, Inc., 1988e)

Title: Acute Toxicity of Anthraquinone to Eastern Oysters (Crassostrea virginica) Under FlowThrough Conditions

This study is: Adequate Not Adequate

This study was found to have following deficiencies: (1) the report did not indicate which 

pesticides were measured analytically in the dilution water and the detection limits for pesticides 

and PCBs were not reported; (2) the measured test substance concentrations at 96 hours were 

approximately 50% lower than concentrations measured at 0 hours indicating that the 

concentration was not maintained over the test period; (3) the flow rate of fresh test solution was 

0.225 L/hour/oyster instead of the flow rate of 1-5 L/hour/oyster recommended in the EPA 

797.1800 test guideline; (4) individual shell growth measurements of each oyster were not 

reported; (5) the water solubility determinations and adequacy of the methods to attain maximum 

test concentrations were not adequately explained; (6) the age of the oysters was not reported; (7) 

oyster growth in controls (2 mm in 96 hours) failed to achieve minimum acceptable shell growth 

levels of 3-4 mm in 96 hours; (8) the lack of a concentration-effect relationship in shell growth 

suggests the reduction in shell deposition may not be a function of the test substance 

concentration; (9) maximum water solubility at the highest test concentration was not achieved 

based on a validated water solubility study conducted after the toxicity test, i.e., the highest test 

concentration of 17 μg/L was approximately 14% of the measured maximum water solubility of 

125 μg/L at a temperature and pH closely approximating the oyster test conditions (22 °C and 

pH of 7.2) if the freshwater and saltwater solubility of the test substance are the same; and (10) 

total organic carbon and particulate levels in the dilution water were not reported. 

TEST SUBSTANCE 

Identity: 9,10-Anthracenedione, CASRN 84-65-1

Remarks: Purity: 99.81% active ingredient (A.I.) according to the study sponsor; 50:50 

mixture of Mobay and ICI anthraquinone powders (Mobay lot #2025865; ICI lot# 611884); 

yellow powder received by the Mobay Corporation.

METHOD

Method/guideline followed: EPA/OTS guidelines (number of guideline not specified)

Test type: Oyster Acute Toxicity Test (flow-through)

GLP compliant?: Yes

Year study was performed: 1988 

Test species: Eastern oyster (Crassostrea virginica) 
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Analytical monitoring: Yes; HPLC

Exposure duration: 96 hours

Concentrations tested: Nominal concentrations: 6.5, 11, 18, 30, and 50 μg a.i./L;

Mean measured concentrations: 6.0, 11, and 17 μg a.i./L at nominal concentrations of 18, 30, 

and 50 μg a.i./L, respectively (measurements at the nominal concentration of 11 μg a.i./L were 

below the detection limit at 96 hours; measurements at the nominal concentration of 6.5 μg a.i./L 

were below the detection limit at 0 and 96 hours)

Test method/conditions remarks: “Forty organisms were exposed in duplicate test aquaria 

(20 per aquaria) in a flow-through system to five concentrations of the test substance, a dilution 

water control and a solvent (acetone) control. Concentrations for this study were based on the 

toxicity of the material to freshwater organisms and the limit of water solubility of anthraquinone 

in freshwater. Prior to initiation of the definitive study, preliminary testing was conducted to 

establish the water solubility under test conditions. Test conditions for this study were generally 

consistent with the requirements published in the EPA ́s Final Test Rule, Parts 704, 795, and 

799, June 1987. Preliminary testing corroborated information provided by the Mobay 

Corporation and established that the limit of water solubility for the test substance in freshwater, 

under the maintained test conditions, was approximately 50 μg a.i./L. Testing performed at SLS 

established that concentrations of anthraquinone equal to or lower than the material's limit of 

water solubility were not toxic to freshwater fish and daphnids. Based on these data, the 

following nominal concentrations were selected for the acute exposure: 50, 30, 18, 11, and 6.5

μg A.I./L Anthraquinone.... Based on the results of the 0- and 96-hour analyses, the mean 

measured test concentrations of the three highest treatment levels were 17, 11, and 6.0 μg a.i./L 

Anthraquinone. Analyses of the fourth treatment level [11 μg a.i./L, nominal] resulted in a 

measured concentration of 5.2 μg a.i./L at 0 hour of the exposure, however, the measurement 

was below the limit of detection established for this study at 96 hours. Analyses of the lowest 

treatment level [6.5 μg a.i./L, nominal] resulted in measured concentrations which were below 

the limit of detection at both 0 and 96 hours.” [Springborn Life Sciences, Inc. 1988e, p. 4-5]

“A continuous flow serial diluter with a dilution factor of 0.60... was used to deliver five test 

concentrations of Anthraquinone, a solvent (acetone) control and a dilution (seawater) water 

control to duplicate test aquaria. The positions of the 14 test aquaria in the water bath were 

determined by random selection. Each glass aquarium measured 60 x 30 x 30 cm and was

equipped with a 10 cm standpipe, which maintained a test solution volume of approximately 18 

L. Exposure aquaria were individually labeled with the nominal concentrations and replicate 

designation. The flow of test solution to each aquarium was 75 mL/minute, which provided 

approximately six volume replacements every 24 hours.” [Springborn Life Sciences, Inc. 1988e, 

p.11] 

Details on Test Solutions: “Prior to the initiation of the definitive exposure, a stock solution 

containing 1.16 mg A.I./mL of the test substance was prepared by dissolving 0.3479 (0.3472 g 

A.I.) in 300 mL of acetone (CAS #67-64-1). This stock was then ultrasonicated for a period of 

approximately 1.5 hours. Due to the limit of water solubility for this test material, the nominal 

test concentration range was lowered and, consequently, the highest nominal treatment level was 

decreased to 50 μg A.I./L. Therefore, the original 1.16-mg A.I./mL stock solution was diluted 
Draft HPV1/HPV2 Chemical Data Adequacy Review (May 2013) CASRN 84-65-1

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50:50 with acetone to a concentration of 0.58 mg A.I./mL. A syringe pump was calibrated to 

deliver 0.0324 mL/minute of the stock solution (0.58 mg A.I./mL) to 375 mL/minute of seawater 

to produce the highest diluter test concentration which was proportionally (60%) diluted to 

produce the test concentration range. The syringe pump also delivered 0.0324 mL/minute of a 

400 μL/mL acetone (in distilled water) stock solution to the solvent control dilution cell. This 

resulted in an acetone concentration in the solvent control aquaria equal to the solvent 

concentration present in the highest test concentration (86 μL acetone/L).” [Springborn Life 

Sciences, Inc. 1988e, p.12-13]

Details on Sampling: “The high, middle, and low test concentrations (A and B replicates) were 

sampled and analyzed for Anthraquinone (A.I.) concentrations one day prior to the initiation of 

the definitive test. Results of these pretest analyses were used to judge whether sufficient 

quantities of test material were being delivered and maintained in the exposure aquaria to initiate 

the definitive study. During the in-life portion of the definitive test, water samples were 

removed at 0 and 96 hours from each replicate solution, including the controls. Upon removal 

from the test aquaria, the exposure solutions were passed through a 0.45 micron filter. At each 

interval, additional samples were taken from both replicate aquaria of the highest test 

concentration that were not filtered before analysis. All samples were removed from the 

approximate mid-point of the aquaria using a glass volumetric pipette. These samples were 

analyzed for the Anthraquinone to confirm the concentration of test material in the exposure 

solutions. Comparison[s] of the analyses of the filtered and unfiltered samples were used to 

establish the presence of insoluble or sorbed test substance. In addition, three quality assurance 

(QA) samples, formulated to test concentrations unknown to the analysts, were prepared at each 

sampling interval and remained with the set of exposure solution samples through the analytical 

process. Results of the analyses of the QA samples were used to judge the precision and quality 

control maintained during the analytical procedures.” [Springborn Life Sciences, Inc. 1988e, p. 

15-16]

Details on Test Organisms: Test organisms “were obtained from Aquacultural Research 

Corporation (ARC), Dennis, Massachusetts where they were reared in natural flowing seawater

from approximately the same source (Massachusetts Bay) as that used as dilution water during 

the toxicity test. During the fourteenth through fourth day prior to testing, measurements made 

at ARC established that the temperature range of the culture water was 19-23°C, the salinity 

range was 28-32%, and the dissolved oxygen concentration range was 87-l00% of saturation. 

Oysters were continuously fed a combination of [two or more species of] marine algae... If any 

oyster appeared unsuitable for testing due to the presence of boring sponges and/or mudworms, it 

was discarded. Oysters were held prior to the test in a wooden epoxy-painted tray through which 

seawater was pumped continuously. The oysters were of similar age and had a mean valve 

height of 42 (± 4) mm (standard deviation).” [Springborn Life Sciences, Inc. 1988e, p.8-9]

“Twenty-four hours prior to testing, 3-5 mm of the new peripheral shell growth of each oyster 

was removed by grinding the shell to a blunt edge using a fine-grit wheel. The test organisms 

were then held overnight, and carefully examined for any signs of stress which might have been 

caused by the removal of shell. Approximately ten oysters, in addition the number required for 

testing, were prepared; any oysters which appeared less than optimal were discarded. 

Immediately prior to the test initiation, the outer shell edge was buffed with an emery board to 
Draft HPV1/HPV2 Chemical Data Adequacy Review (May 2013) CASRN 84-65-1

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remove any new shell deposition. The exposure of oysters was initiated by impartially selecting 

and placing 20 oysters in each test aquarium (40 per treatment).” [Springborn Life Sciences, Inc.

1988e, p.13]

“Biological observations were made daily during the exposure in order to detect any mortality of 

oysters and to record any visible abnormalities, such as excessive mucus production or a failure 

to siphon and feed, as evidenced by a lack of feces and pseudofeces production. After 96 hours 

of exposure the oysters were removed and the new shell growth was measured microscopically 

to the nearest 0.1 mm using a calibrated micrometer.” [Springborn Life Sciences, Inc. 1988e, p. 

14]

The test temperature was 19-20°C, the pH was 7.8-8.0, the dissolved oxygen concentration was 

6.7-7.6 mg/L, and the salinity was 32 ppt. Reported results are based on measured 

concentrations of the test substance in the exposure vessels. 

RESULTS

Endpoint value(s): 96-hour EC50 > 17 μg a.i./L (mean measured concentration) based on the 

reduction in shell deposition.

Results remarks: During the exposure period, undissolved material was observed at the tip of 

the syringe tubing used to deliver stock solution of test substance to the diluter chemical cells. 

Throughout the 96-hour test, no undissolved material (e.g., precipitate, film on test solution 

surface), was observed in the diluter cells or test aquaria. The measured concentrations of the 

replicates (A/B) at 0 hours were <LOD/<LOD, <LOD/<LOD, <LOD/<LOD, 4.7/5.7, 9.7/8.0, 

14/14, and 22/21 μg a.i./L at nominal concentrations of 0 (control), 0 (solvent control), 6.5, 11, 

18, 30, and 50 μg a.i./L, respectively. At 96 hours, the measured concentrations were 

<LOD/<LOD, <LOD/<LOD, <LOD/<LOD, <LOD/<LOD, 3.7/2.9, 8.3/7.1, and 11/14 μg a.i./L 

at nominal concentrations of 0 (control), 0 (solvent control), 6.5, 11, 18, 30, and 50 μg a.i./L, 

respectively, indicating an approximate 50% decrease in concentration for the duration of the 

test. The limit of detection (LOD) was 3.0 μg a.i./L at 0 hours and 2.8 μg a.i./L at 96 hours.

“During the 96-hour exposure, oysters in all exposure solutions and the controls appeared to be 

feeding and siphoning normally, and no mortality occurred. Comparison of biological response 

data did not establish a concentration-effect relationship within the concentration range tested. 

At the end of the exposure, the mean shell deposition of oysters at all exposure levels tested was 

slightly (15-25%) but significantly (P = ≤ 0.05) reduced in comparison to the pooled (dilution 

water and solvent control) control organisms. Shell deposition among oysters exposed to the 

highest mean measured test concentration of Anthraquinone (17 μg A.I./L) was reduced 15% as 

compared to the pooled controls.” [Springborn Life Sciences, Inc 1988e, p. 20]

Percent reduction of shell deposition, compared to pooled controls, was 20, 25, 15, 15, and 15% 

at nominal concentrations of 6.5, 11, 18, 30, and 50 μg a.i./L, respectively.

The EC50 for this study was empirically estimated to be > 17 μg a.i./L, the highest concentration 

of anthraquinone tested.
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CONCLUSIONS

“Based on this [sic] data, it was established that under the conditions maintained during this 

study (e.g., water quality, solution temperature, solvent concentration, mixing period),

Anthraquinone is not acutely toxic to Eastern oysters at and below the materials’ limit of water 

solubility.” [Springborn Life Sciences, Inc 1988e, p. 20] 

STUDY RELIABILITY

[3] Not reliable. This study was submitted in response to EPA’s 1987 test rule for CASRN 84-

65-1 (Anthraquinone; Final Reporting and Recordkeeping Requirements and Test Rule; 52 FR 

21018, June 4, 1987) and was found to have following deficiencies: (1) the report did not 

indicate which pesticides were measured analytically in the dilution water and the detection 

limits for pesticides and PCBs were not reported; (2) the measured test substance concentrations

at 96 hours were approximately 50% lower than concentrations measured at 0 hours indicating 

that the concentration was not maintained over the test period; (3) the flow rate of fresh test 

solution was 0.225 L/hour/oyster instead of the flow rate of 1-5 L/hour/oyster recommended in 

the EPA 797.1800 test guideline; (4) individual shell growth measurements of each oyster were 

not reported; (5) the water solubility determinations and adequacy of the methods to attain 

maximum test concentrations were not adequately explained; (6) the age of the oysters was not 

reported; (7) oyster growth in controls (2 mm in 96 hours) failed to achieve minimum acceptable 

shell growth levels of 3-4 mm in 96 hours; (8) the lack of a concentration-effect relationship in 

shell growth suggests the reduction in shell deposition may not be a function of the test substance 

concentration; (9) maximum water solubility at the highest test concentration was not achieved 

based on a validated water solubility study conducted after the toxicity test, i.e., the highest test 

concentration of 17 μg/L was approximately 14% of the measured maximum water solubility of 

125 μg/L at a temperature and pH closely approximating the oyster test conditions (22 °C and 

pH of 7.2) if the freshwater and saltwater solubility of the test substance are the same; and (10) 

total organic carbon and particulate levels in the dilution water were not reported.

REFERENCES

Springborn Life Sciences, Inc. 1988e. Acute Toxicity of Anthraquinone to Eastern Oysters 

(Crassostrea virginica) Under Flow-Through Conditions. Report # 88-12-2883. SLS Study 

#274.0488.6177.504. Sponsor: Mobay Corporation. 

Text in quotes is taken directly from Springborn Life Sciences, Inc., 1988e.
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C-3. Toxicity to Aquatic Plants

Summary of endpoint: Not adequate. Although no adequate algal toxicity studies are 

available, the toxicity to aquatic plants endpoint is not considered to be a data gap at this time, 

given that: (1) low toxicity has been observed in fish and daphnids (i.e., no adverse effects at or 

near the limit of water solubility); and (2) according to EPA’s 1987 test rule for CASRN 84-65-1 

(Anthraquinone; Final Reporting and Recordkeeping Requirements and Test Rule; 52 FR 21018, 

June 4, 1987), “sufficient data are available from the study done by Chillingworth [1974]... to 

reasonably predict anthraquinone’s toxicity to algae”. There is no need for further testing of the 

toxicity to aquatic plants endpoint at this time.

Study 1: Toxicity to Selenastrum capricornutum (Chillingworth, 1974)1

Title: The toxicity of aminoanthraquinone dyes to fish and algae

This study is: Adequate Not Adequate

This study is not adequate because: (1) Purity and composition of the test substance were not 

provided; (2) Test substance concentrations in growth media were not analytically monitored; 

(3) Nominal loading concentrations greatly exceeded the measured water solubility limit; (4) pH 

was not measured in growth media over the course of the study; (5) One component of the 

growth medium, FeCl3, may have been omitted; (6) Quality parameters were not provided for the 

water used to prepare the growth medium; (7) The solubility of 9,10-anthracenedione in the 

growth medium was not determined; (8) Light intensity was not specified and was not measured; 

(9) Whereas the duration of a study of Selenastrum capricornutum is typically 96 hours, the 

duration of this study was 14 days and biomass measurements (cells/mL) were performed in this 

study only at days 7 and 14 (ASTM E1218-04 strongly recommends that biomass measurements 

be performed daily, but minimally at the beginning and end of the test, and the Algal Assay 

Procedure Bottle Test guideline [NERP/EPA, 1971], which the Chillingworth [1974] study is 

intended to follow, states that “it is necessary that measurements of biomass be made at least 

daily during the first 5 days of incubation” to determine the maximum specific growth rate); and 

(10) Since growth was assessed only at days 7 and 14 and cell density at day 14 was only about 

3-fold greater than at day 7, growth had nearly reached a plateau by day 7 and exponential 

growth was not maintained throughout the test; consequently, potential effects of 

9,10-anthracenedione on growth rate (e.g., slower growth during the initial 96 hours of exposure, 

prior to nutrient depletion) may not have been detected.

TEST SUBSTANCE 

Identity: 9,10-Anthracenedione, CASRN 84-65-1 (test substance referred to as Anthraquinone 

in study report)

 

1 In an email dated March 16, 2012, Chemical Products Corporation requested that EPA consider data from 

Chillingworth (1974) to satisfy the C6 testing requirement for 9,10-anthracenedione.
Draft HPV1/HPV2 Chemical Data Adequacy Review (May 2013) CASRN 84-65-1

41

Remarks: Purity not indicated; the only relevant information was that anthraquinone was 

“made by Eastman” 

METHOD

Method/guideline followed: Algal Assay Procedure Bottle Test (NERP/EPA, 1971) 

Test type: Alga, Growth Inhibition Test

GLP compliant?: Pre-GLP

Year study was performed: 1974

Test species: Green algae (Selenastrum capricornutum); source: Thomas E. Maloney of the 

National Eutrophication Research Program

Analytical monitoring: No

Exposure duration: 7 and 14 days 

Concentrations tested: Nominal concentrations: 0 (control), 1, and 10 mg/L. 

Test method/conditions remarks: The test was conducted at concentrations above the reported 

water solubility limit values of 74.6 μg/L (20°C, pH 7; Case Consulting Laboratories, 20061

) and 

1.35 mg/L (25 °C; Yalkowsky and Dannenfelser, 19902). 

There is uncertainty that the maintenance and growth medium used in this study included the 

FeCl3 that is present in the Synthetic Algal Nutrient Medium described by NERP/EPA (1971) 

and the freshwater algae medium described in ASTM E1218-04. Otherwise, the culture medium 

contained the macro- and micro-nutrients specified in NERP/EPA (1971). The source and 

quality of the water used for growth medium preparation were not indicated; NERP/EPA (1971) 

specifies the use of glass-distilled water and ASTM specifies the use of ASTM Type I water.

Triplicate flasks were prepared for controls and each of the two test concentrations. Each 

500-mL flask contained 100 mL of test solution and the tops of the flasks were covered with 

aluminum foil. Incubator temperature was 24 ± 1°C with constant illumination from fluorescent 

bulbs. Light intensity (lumens/m2

) was not specified. The initial pH value of the growth 

medium was not specified and pH was not measured over the course of the study. The initial 

 

1 Case Consulting Laboratories, Inc. 2006. Physical and Chemical Characteristics of Anthraquinone: 

Octanol/Water Partition Coefficient and Solubility. Sponsor: Chemical Products Technologies, LLC. Study No. 

3320-02. November 20, 2006. 2 Yalkowsky, S.H and Dannenfelser, R.M. 1990. AQUASOL Database of Aqueous Solubility. Fifth edition. 

Tucson, AZ: University of Arizona, College of Pharmacy. Current book version: Yalkowsky, F.H. and He, Y. 

2003. Handbook of Aqueous Solubility Data. CRC Press, Boca Raton, FL (ISBN 0-8493-1532-8). Value obtained 

from EPI Suite v4.1.
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concentration of algae was 103 cells/mL. For each flask, cell density measurements (cells/mL; 

surrogate for biomass) were performed in duplicate at days 7 and 14. Additional details were not 

given.

RESULTS

Endpoint value(s): 7- and 14-day EC50 (biomass) > 10 mg/L; 7- and 14-day NOEC (biomass) > 

10 mg/L

 

Results remarks: Mean cell densities at day 7 were 2.43 x 106, 2.61 x 106, and 2.46 x 106

cells/mL at test concentrations of 0, 1, and 10 mg 9,10-anthracenedione/L, respectively. At 

day 7, the ratios of cell densities in treated:control flasks were 1.074 at 1 mg/L and 1.012 at 10 

mg/L. Mean cell densities at day 14 were 6.91 x 106, 8.13 x 106, and 7.03 x 106 cells/mL at test 

concentrations of 0, 1, and 10 mg/L, respectively. At day 14, the ratios of cell densities in 

treated:control flasks were 1.177 at 1 mg/L and 1.017 at 10 mg/L. Cell counts in controls 

increased by a factor of approximately 2400 during the first 7 days in culture.

The report did not indicate whether or not insoluble material was noted during the test.

CONCLUSIONS

The 7- and 14-day EC50 and NOEC values for inhibition of cell density of Selenastrum 

capricornutum were > 10 mg/L.

STUDY RELIABILITY

[3] Not reliable. The study had the following deficiencies compared with ASTM E1218-04 

guidance: (1) Purity and composition of the test substance were not provided; (2) Test substance 

concentrations in growth media were not analytically monitored; (3) Nominal loading 

concentrations greatly exceeded the measured water solubility limit; (4) pH was not measured in 

growth media over the course of the study; (5) One component of the growth medium, FeCl3, 

may have been omitted; (6) Quality parameters were not provided for the water used to prepare

the growth medium; (7) The solubility of 9,10-anthracenedione in the growth medium was not 

determined; (8) Light intensity was not specified and was not measured; (9) Whereas the 

duration of a study of Selenastrum capricornutum is typically 96 hours, the duration of this study 

was 14 days and biomass measurements (cells/mL) were performed in this study only at days 7 

and 14 (ASTM E1218-04 strongly recommends that biomass measurements be performed daily, 

but minimally at the beginning and end of the test, and the Algal Assay Procedure Bottle Test 

guideline [NERP/EPA, 1971], which the Chillingworth [1974] study is intended to follow, states 

that “it is necessary that measurements of biomass be made at least daily during the first 5 days 

of incubation” to determine the maximum specific growth rate); and (10) Since growth was 

assessed only at days 7 and 14 and cell density at day 14 was only about 3-fold greater than at 

day 7, growth had nearly reached a plateau by day 7 and exponential growth was not maintained 

throughout the test; consequently, potential effects of 9,10-anthracenedione on growth rate (e.g., 

slower growth during the initial 96 hours of exposure, prior to nutrient depletion) may not have 

been detected. 
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REFERENCES

Chillingworth, M.A. 1974. The toxicity of aminoanthraquinone dyes to fish and algae. 

M.S.P.H. Thesis, Dept. Environ. Sci. Eng., Univ. North Carolina, Chapel Hill. 

NERP/EPA. National Eutrophication Research Program/Environmental Protection Agency. 

1971. Algal Assay Procedure Bottle Test, August, 1971.
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Study 2: Toxicity to Selenastrum capricornutum (Giddings, 1979)

Title: Acute toxicity to Selenastrum capricornutum of Aromatic Compounds from Coal 

Conversion

This study is: Adequate Not Adequate

The study had the following deficiencies compared with the ASTM E1218-04 guideline: (1) the 

duration of this study was 4 hours, whereas the duration of a study of Selenastrum 

capricornutum is typically 96 hours; (2) purity and composition of the test substance were not 

provided; (3) test substance concentrations in growth media were not analytically monitored; (4) 

pH was not measured in growth media over the course of the study; (5) quality parameters were 

not provided for the water used to prepare the growth medium; and (6) light intensity was not 

specified and was not measured.

TEST SUBSTANCE 

Identity: Supporting Compound, 1,4-Anthraquinone

Remarks: Reagent grade (purity not specified)

METHOD

Method/guideline followed: Not reported 

Test type: Alga, Growth Inhibition Test

GLP compliant?: Pre-GLP

Year study was performed: Not reported

Test species: Green algae (Selenastrum capricornutum); source: EPA National Environmental 

Research Laboratory at Corvallis, Oregon

Analytical monitoring: No

Exposure duration: 4 hours 

Concentrations tested: Nominal concentrations: 1, 10 and 100% saturation.

Test method/conditions remarks: “For compounds whose solubility was less than 1000 

mg/mL, saturated solutions were prepared by adding excess compound to sterile medium and 

stirring overnight; the solutions were then filtered (Whatman #41 filter paper) and diluted 

approximately. These compounds were then tested at 1, 10, and 100% saturation.” [Giddings 

1979, p. 361]
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1,4-Anthraquinone “was tested at three concentrations, with two replicates per concentration. 

Three control bottles, without toxicant, were included in each experiment. Carbon-14 fixation in 

the test solutions was expressed as a percentage of the mean of the controls” [Giddings 1979, p. 

362]

“BOD bottles (125-mL) were filled with toxicant solutions prepared as described above. One 

mL was then removed from each bottle to leave room for the algal suspension. A portion of a 

rapidly-growing S. capricornutum culture was concentrated by centrifugation (1500 rpm, 25 

min) to approximately 1.25 x 107 cells/mL, and 1 mL of this suspension was added to each test 

bottle. The bottles were placed on a rotary shaker at 100 rpm in an environmental chamber 

(24°C...). After 2 h, 0.2 mL of an NaH14CO3 solution (10 μCi/mL...) was added to each bottle. 

After an additional 2 h incubation, biological activity was stopped by the addition of 0.3 mL 

formalin” [Giddings 1979, p. 361]

“Photosynthesis was determined by measurement of fixed 14C as follows. Two 5-mL samples 

were taken from each bottle and placed in 20-mL liquid scintillation vials. Each sample was 

acidified with 0.1 mL of 0.1 N HCl and bubbled with air for 20 min to remove inorganic 

carbon... Each vial then received 15 mL Aquasol..., and the activity remaining in the samples

was counted.” [Giddings 1979, p. 362]

RESULTS

Endpoint value(s): 4-hour EC50 > 100% saturation (based on inhibition of photosynthesis)

 

Results remarks: Carbon-14 fixation (measurement of photosynthesis) by S. capricornutum

after 4 hours of exposure was as follows: 85% of control at 100% saturation of test substance; 

91% of control at 10% saturation of test substance; 97% of control at 1% saturation of test 

substance. Therefore, a concentration-related decrease in photosynthesis was observed. 

However, inhibition of photosynthesis was only 15% in a saturated test solution.

“Anthraquinone, presumably due to its insolubility, was essentially non-toxic to S. 

capricornutum...” [Giddings 1979, p. 362]

CONCLUSIONS

Anthraquinone had low toxicity in S. capricornutum.

STUDY RELIABILITY

[3] Not reliable. The study had the following deficiencies compared with the ASTM E1218-04 

guideline: (1) the duration of this study was 4 hours, whereas the duration of a study of 

Selenastrum capricornutum is typically 96 hours; (2) purity and composition of the test 

substance were not provided; (3) test substance concentrations in growth media were not 

analytically monitored; (4) pH was not measured in growth media over the course of the study; 

(5) quality parameters were not provided for the water used to prepare the growth medium; and 

(6) light intensity was not specified and was not measured. 
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REFERENCES

Giddings. J.M. 1979. Acute toxicity to Selenastrum capricornutum of aromatic compounds from 

coal conversion. Bull. Environ. Contam. Toxicol. 23: 360-364.

Text in quotes is taken verbatim from Giddings, 1979.
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C-4. Acute Toxicity 

Summary of endpoint: Adequate

Study 1: Acute oral toxicity in rats (From ECHA website, Exp Key Acute toxicity: oral.001)

Title: Not provided

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: 9,10-Anthracenedione, CASRN 84-65-1

Remarks: Purity: 98.9 wt % (main component) other significant impurities include anthracene 

0.01 wt %, 2,3-Naphthalic anhydride 0.25%, Naphtho[2,3-b]thiphenquinone 0.33%, 

1,4-Anthraquinone 0.32%; Batch No. V 1161; Appearance: yellow crystalline powder

METHOD

Method/guideline followed: EU Method B.1 tris (Acute Oral Toxicity – Acute Toxic Class 

Method)

Test type: Acute Oral Toxicity

GLP compliant?: Yes

Year study performed: 2009

Test species: Wistar rats

Number of animals/sex/dose: 6 females

Route of administration: Gavage

Vehicle: Olive oil

Doses/concentrations tested: 2000 mg/kg-bw

Statistical methods used: None stated

Test method/conditions remarks: The test substance was administered in a single dose as 

solution in vehicle (olive oil), given orally via gavage to two groups of three female Wistar rats

at a dosing volume of 1 ml/100 g of animal body weight. The dosing was performed 

sequentially in two groups of three females: group No. 1 – first step using the starting dose of 

2000 mg/kg of body weight and group No.2 – second step using the same dose. Animals were 
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observed for 14 days following dosing. Body weight was measured before application, on the 8th

day of the study and at day 15, before euthanasia of animals. Observations for mortality and 

clinical examinations were performed daily. Gross necropsy of survivors was performed at study 

termination.

Test animals

-Source: breeding farm VELAZ s.r.o., Koleč u Kladna, Czech Republic

-Age at study initiation: 8-10 weeks at time of application

-Housing: animal room with monitoring conditions – 3 animals of one sex in one plastic 

breeding cage

-Diet: ST 1 BERGMAN – standard pelleted diet ad libitum

-Fasting period before study: about 20 hours

-Water: drinking tap water ad libitum

Acclimation period: 5 days

Environmental conditions

-Temperature (°C): room temperature 22 +/- 3°C, permanently monitored

-Humidity (%): relative humidity 30 – 70 %, permanently monitored

-Air changes (per hr): approximately 15

-Photoperiod (hrs dark / hrs light): 12/12

RESULTS

Endpoint value(s): LD50 (female) > 2000 mg/kg-bw

Results remarks: The test substance administered at the dose of 2000 mg/kg caused no death of 

animals. No clinical signs of intoxication were observed in all animals. No pathologic 

macroscopic changes were diagnosed during pathological examination in all animals. According 

to the study results, the LD50 of the test substance for female rats is higher than 2000 mg/kg-bw. 

The classification of the test substance toxicity was performed according to the Directive 

67/548/EEC, Annex VI. Part 3.1.5 and 3.2. Based on the test results and according to the EC 

criteria for classification and labeling requirements for dangerous substances and preparations, 

the test substance did not fall into any of the quoted categories of toxicity and has no obligatory 

labeling requirement in this respect.

CONCLUSIONS

The test substance was evaluated on the basis of mortality, clinical signs of intoxication, body 

weight increments during the observation period and necropsy findings at the end of study. The 

test substance administered at the dose of 2000 mg/kg caused no death of animals. No clinical 

signs of intoxication were observed in all animals. No pathologic macroscopic changes were 

diagnosed during pathological examination in all animals.
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STUDY RELIABILITY

[2] Reliable with restrictions. The summary provided limited information on the study methods 

and results and did not include a full citation for the study report. 

REFERENCES

Study available from ECHA website, http://echa.europa.eu, first row of the “Search results” table 

resulting from a “Chemical Substance Search” for CASRN 84-65-1. Study indicated as Exp Key 

Acute toxicity: oral.001. The data source is referred to as a “study report”, with a report date of 

2009-07-23. No other reference information is provided.

Copyright Notice: © European Chemicals Agency, 2007-2011. Reproduction is authorised 

provided the source is fully acknowledged in the form "Source: European Chemicals Agency, 

http://echa.europa.eu/", and provided written notification is given to the ECHA Communication 

Unit (info (at) echa.europa.eu).
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Study 2: Acute inhalation toxicity in rats (From ECHA website, Exp Key Acute toxicity: 

inhalation.001)

Title: Not provided

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: 9,10-Anthracenedione, CASRN 84-65-1

Remarks: Not provided

METHOD

Method/guideline followed: Not stated

Test type: Acute Inhalation Toxicity

GLP compliant?: Pre-GLP

Year study performed: 1974-1975

Test species: BLU: (LE) rats

Number of animals/sex/dose: 5

Route of administration: Inhalation (dust; whole body)

Vehicle: Clean air

Doses/concentrations tested: 244.0 mg/m3 (0.244 mg/L; maximum attainable concentration)

Statistical methods used: Not stated

Test method/conditions remarks: The dust was generated by passing a stream of clean, dry air 

(-40°C dew-point) through a Ferris wheel generator containing the undiluted test material and 

into the test chamber. A 4-hour exposure was followed by a 14-day observation period.

RESULTS

Endpoint value(s): LC50 > 244 mg/m3 (0.244 mg/L)

Results remarks: There were no untoward reactions during exposure or the 14-day observation 

period which followed. The average two-week body weight gains were within the normal limits. 
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Necropsy, performed on all rats at the end of the observation period, did not reveal any gross 

pathologic alterations.

CONCLUSIONS

None provided

STUDY RELIABILITY

[2] Reliable with restrictions. The summary provided limited information on the study methods 

and results and did not include a full citation for the study report. 

REFERENCES

Study available from ECHA website, http://echa.europa.eu, first row of the “Search results” table 

resulting from a “Chemical Substance Search” for CASRN 84-65-1. Study indicated as Exp Key 

Acute toxicity: inhalation.001. The data source is referred to as a “study report”, with a report 

date of 1975-02-25. No other reference information is provided.

Copyright Notice: © European Chemicals Agency, 2007-2011. Reproduction is authorised 

provided the source is fully acknowledged in the form "Source: European Chemicals Agency, 

http://echa.europa.eu/", and provided written notification is given to the ECHA Communication 

Unit (info (at) echa.europa.eu).
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C-5. Repeated-Dose Toxicity

Summary of endpoint: Adequate

Study 1: 14-Week repeated-dose oral toxicity study in rats (NTP, 2005)

Title: Toxicology and carcinogenesis studies of anthraquinone (CAS No. 84-65-1) in F344/N 

rats and B6C3F1 mice (feed studies) 

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: 9,10-Anthracenedione, CASRN 84-65-1

Remarks: 99.8% pure

METHOD

Method/guideline followed: NTP Protocols (Experimental design presented in Table 1 of NTP 

[2005]).

Test type: 14-Week Oral Toxicity Study in Rats 

GLP compliant?: Yes

Year study performed: 1994

Test species: Fischer 344 rats (male and female)

Number of animals/sex/dose: 20

Route of administration: Oral – dietary

Vehicle: None

Exposure duration: 14 weeks

Doses/concentrations tested: 0, 1875, 3750, 7500, 15,000, and 30,000 ppm (equivalent to 

approximately 135, 275, 555, 1130, and 2350 mg/kg-bw/day)

Statistical methods used: “Survival Analyses: The probability of survival was estimated by the

product-limit procedure of Kaplan and Meier (1958) and is presented in the form of graphs. 

Animals found dead of other than natural causes or missing were censored from the survival 

analyses; animals dying from natural causes were not censored. Statistical analyses for possible 

dose-related effects on survival used Cox’s (1972) method for testing two groups for equality 
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and Tarone’s (1975) life table test to identify dose-related trends. All reported P values for the

survival analyses are two sided.”

“Calculation of Incidence: The incidences of neoplasms or nonneoplastic lesions are 

presented... as the numbers of animals bearing such lesions at a specific anatomic site and the 

numbers of animals with that site examined microscopically. For calculation of statistical 

significance, the incidences of most neoplasms... and all nonneoplastic lesions are given as the 

numbers of animals affected at each site examined microscopically. However, when 

macroscopic examination was required to detect neoplasms in certain tissues (e.g., harderian

gland, intestine, mammary gland, and skin) before microscopic evaluation, or when neoplasms 

had multiple potential sites of occurrence (e.g., leukemia or lymphoma), the denominators 

consist of the number of animals on which a necropsy was performed.” Survival-adjusted

neoplasm rates for each group and each site-specific neoplasm are also presented. The “survivaladjusted rate (based on the Poly-3 method described below) accounts for differential mortality by 

assigning a reduced risk of neoplasm, proportional to the third power of the fraction of time on 

study, to animals that do not reach terminal sacrifice.”

“Analysis of Neoplasm and Nonneoplastic Lesion Incidences: The Poly-k test (Bailer and 

Portier, 1988; Portier and Bailer, 1989; Piegorsch and Bailer, 1997) was used to assess neoplasm 

and nonneoplastic lesion prevalence. This test is a survival-adjusted quantal-response procedure

that modifies the Cochran-Armitage linear trend test to take survival differences into account. 

More specifically, this method modifies the denominator in the quantal estimate of lesion 

incidence to approximate more closely the total number of animal years at risk. For analysis of a 

given site, each animal is assigned a risk weight. This value is one if the animal had a lesion at 

that site or if it survived until terminal sacrifice; if the animal died prior to terminal sacrifice and 

did not have a lesion at that site, its risk weight is the fraction of the entire study time that it 

survived, raised to the kth power. This method yields a lesion prevalence rate that depends only 

upon the choice of a shape parameter for a Weibull hazard function describing cumulative lesion

incidence over time (Bailer and Portier, 1988). Unless otherwise specified, a value of k=3 was 

used in the analysis of site-specific lesions. This value was recommended by Bailer and Portier 

(1988) following an evaluation of neoplasm onset time distributions for a variety of site-specific 

neoplasms in control F344 rats and B6C3F1 mice (Portier et al., 1986). Bailer and Portier 

(1988) showed that the Poly-3 test gave valid results if the true value of k was anywhere in the 

range from 1 to 5. A further advantage of the Poly-3 method is that it does not require lesion 

lethality assumptions. Variation introduced by the use of risk weights, which reflect differential 

mortality, was accommodated by adjusting the variance of the Poly-3 statistic as recommended

by Bieler and Williams (1993). Tests of significance included pairwise comparisons of each 

exposed group with controls and a test for an overall exposure-related trend. Continuitycorrected Poly-3 tests were used in the analysis of lesion incidence, and reported P values are 

one sided... For neoplasms and nonneoplastic lesions detected at the interim evaluations, the 

Fisher exact test (Gart et al., 1979), a procedure based on the overall proportion of affected

animals, was used.”

“Analysis of Continuous Variables: Two approaches were employed to assess the significance

of pairwise comparisons between exposed and control groups in the analysis of continuous 

variables. Organ and body weight data, which historically have approximately normal 
Draft HPV1/HPV2 Chemical Data Adequacy Review (May 2013) CASRN 84-65-1

54

distributions, were analyzed with the parametric multiple comparison procedures of Dunnett 

(1955) and Williams (1971, 1972). Soluble protein and α-2u-globulin concentrations were 

analyzed by Dunnett’s one-tailed t-test for comparing means to the 0 ppm group and Duncan’s 

multiple range test for comparing all means to each other. Hematology, clinical chemistry, 

urinalysis, toxicokinetic, spermatid, and epididymal spermatozoal data, which have typically

skewed distributions, were analyzed using the nonparametric multiple comparison methods of 

Shirley (1977) (as modified by Williams, 1986) and Dunn (1964). Jonckheere’s test 

(Jonckheere, 1954) was used to assess the significance of the dose-related trends and to 

determine whether a trend-sensitive test (Williams’ or Shirley’s test) was more appropriate for 

pairwise comparisons than a test that does not assume a monotonic dose-related trend (Dunnett’s 

or Dunn’s test). Prior to statistical analysis, extreme values identified by the outlier test of Dixon 

and Massey (1957) were examined by NTP personnel, and implausible values were eliminated 

from the analysis. Average severity values were analyzed for significance with the MannWhitney U test (Hollander and Wolfe, 1973). Because vaginal cytology data are proportions (the 

proportion of the observation period that an animal was in a given estrous stage), an arcsine 

transformation was used to bring the data into closer conformance with a normality assumption. 

Treatment effects were investigated by applying a multivariate analysis of variance (Morrison,

1976) to the transformed data to test for simultaneous equality of measurements across exposure

concentrations.”

Test method/conditions remarks: “Male and female F344/N rats... were obtained from 

Taconic Farms (Germantown, NY). On receipt, rats... were approximately 4 weeks old. Rats 

were quarantined for 11 (males) or 12 (females) days...; rats... were approximately

6 to 7 weeks old on the first day of the studies. Before initiation of the studies, five male and 

five female rats... were randomly selected for parasite evaluation and gross observation for 

evidence of disease. Serologic analyses were performed on up to five male and five female 

sentinel rats... 1 month after study start and on up to five male and five female control rats... at 

study termination using the protocols of the NTP Sentinel Animal Program...”

Groups of 10 male and 10 female rats were administered diets containing 0, 1875, 3750, 7500, 

15,000, or 30,000 ppm (equivalent to approximately 135, 275, 555, 1130, and 2350 mg/kgbw/day) of anthraquinone for 14 weeks. “Clinical pathology study groups of 10 males and 10 

females received the same concentrations of anthraquinone for 25 days. Water was available ad 

libitum and feed was available ad libitum except during urine collection periods. Rats were 

housed five per cage... Clinical findings were recorded and animals were weighed weekly and 

at the end of the studies. Feed consumption was recorded twice weekly...”

“Blood was collected from the retroorbital sinus of clinical pathology study rats under carbon 

dioxide anesthesia on days 4 and 22. Using the same method, blood was collected from all core 

study rats... surviving to the end of the studies for hematology and clinical chemistry...

analyses. Blood samples for hematology analyses were placed into microcollection tubes 

containing potassium EDTA. Erythrocyte, platelet, and leukocyte counts, hematocrit values,

hemoglobin concentration, mean cell volume, mean cell hemoglobin, and mean cell hemoglobin 

concentration were determined using a Serono-Baker System 9000 hematology analyzer 

(Serono-Baker Diagnostics, Allentown, PA). Differential leukocyte counts and erythrocyte and 

platelet morphologies were determined microscopically from blood smears stained with Wright-
Draft HPV1/HPV2 Chemical Data Adequacy Review (May 2013) CASRN 84-65-1

55

Giemsa stain on a Hema-Tek slide stainer (Miles Laboratory, Ames Division, Elkhart, IN). A

Miller disc was used to determine reticulocyte counts from smears prepared with blood stained 

with new methylene blue. For clinical chemistry analyses, blood samples were placed into 

microcollection serum separator tubes, centrifuged, and the serum samples were analyzed using a 

Hitachi 704R chemistry analyzer (Boehringer Mannheim, Indianapolis, IN) using commercially 

available reagents.” The following hematology parameters were measured: erythrocyte, 

reticulocyte, and platelet counts; hematocrit values; hemoglobin concentration; erythrocyte and

platelet morphology; mean cell volume; mean cell hemoglobin; mean cell hemoglobin 

concentration; and leukocyte count and differentials. The following clinical chemistry 

parameters were measured: urea nitrogen, creatinine, total protein, albumin, alanine 

aminotransferase, alkaline phosphatase, creatine kinase, sorbitol dehydrogenase, and bile salts. 

“Urine samples were collected from clinical pathology study rats on days 8 and 26 and from core 

study rats on day 89. All rats were placed in metabolism cages for 16 hours, and urine collection 

tubes were placed in an ice bath during collection. Clinical pathology study rats were discarded 

without necropsy following the day 26 urine collection period. Urine total volume was

measured, and specific gravity was determined using an American Optical Refractometer/Total

Solids Meter (American Optical, Buffalo, NY). All other urine parameters were determined 

using a Hitachi 704R analyzer.” The following urine parameters were measured: creatinine, 

glucose, total protein, aspartate aminotransferase, N-acetyl-β-D-glucosaminidase,

γ-glutamyltransferase, total volume, and specific gravity. 

“At the end of the 14-week studies, samples were collected for sperm motility evaluations on 

core study rats exposed to 0, 7500, 15,000, and 30,000 ppm.” “The following parameters were 

evaluated: spermatid heads per testis and per gram testis, spermatid counts, and epididymal 

spermatozoal motility and concentration. The left cauda epididymis, left epididymis, and left 

testis were weighed. Vaginal samples were collected for up to 12 consecutive days prior to the 

end of the studies from core study 0, 7500, 15,000, and 30,000 ppm female rats and mice for 

vaginal cytology evaluations. The following parameters were evaluated: estrous cycle length 

and relative frequency of the estrous stages.” 

“Necropsies were performed on all core study animals. The heart, right kidney, liver, lungs, 

right testis, and thymus were weighed. Tissues for microscopic examination were fixed and 

preserved in 10% neutral buffered formalin, processed and trimmed, embedded in paraffin, 

sectioned to a thickness of 4 to 6 μm, and stained with hematoxylin and eosin. Complete 

histopathologic examinations were performed on all core study 0 and 30,000 ppm rats...” “In 

addition to gross lesions and tissue masses, the following tissues [and organs] were examined

[microscopically]: adrenal gland, bone with marrow, brain, clitoral gland, esophagus,... heart 

and aorta, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), 

kidney, liver, lung and mainstem bronchi, lymph nodes (mandibular and mesenteric), mammary 

gland (with adjacent skin), nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial 

gland, prostate gland, salivary gland, spleen, stomach (forestomach and glandular), testis (with 

epididymis and seminal vesicle), thymus, thyroid gland, trachea, urinary bladder, and uterus. In 

addition, the bone marrow, liver, kidney, spleen, and thyroid gland of male and female rats, [and]

the urinary bladder of female rats... were examined in all lower exposure groups.” 
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56

“At necropsy, right kidneys of male rats were bisected transversely. The caudal half was placed 

in a vial containing phosphate-buffered saline and frozen. Before analysis, each sample was 

defrosted, the buffer was removed and replaced with a sodium/potassium buffer, the sample was 

homogenized, and the supernatant was drawn off. Soluble protein content was measured in a 

1:50 dilution with phosphate-buffered saline/Tween using a pyrogallol assay. Concentrations of 

α-2u-globulin were measured using a validated enzyme-linked immunosorbent assay (Fuciarelli 

et al., 1996).”

Endpoint value(s): LOAEL = 1875 ppm (approximately 135 mg/kg-bw/day) based on 

decreased body weights in females; increased liver and kidney weights (both sexes); responsive 

anemia (both sexes); liver hypertrophy (both sexes); eosinophilic hyaline droplets in the kidney 

(both sexes); congestion, hematopoietic cell proliferation, and pigmentation of the spleen (both 

sexes); increased levels of serum total protein and albumin (both sexes); increased levels of 

serum creatinine (males); decreased serum bile salt levels (males); increased concentrations of 

urine protein and glucose (males) and increased activities of urine aspartate aminotransferase and 

N-acetyl-β-D-glucosaminidase (both sexes); and bone marrow hyperplasia in females. A 

NOAEL was not established.

Results remarks: All rats survived to scheduled termination. Final mean body weights and 

body weight changes of all exposed female groups were less than in the controls. Absolute and 

relative liver and kidney weights of exposed groups of males and females were greater than in 

controls. In all exposed groups, mean absolute testes weight was increased compared with 

controls; relative testes weight was also increased at ≥ 7500 ppm. Responsive anemia was 

present in all exposed groups of males and females at the end of the study. Serum total protein

and albumin levels were increased and serum alkaline phosphatase activity was decreased in all 

exposed groups of males and females. At the end of the study, an increase in serum creatinine 

levels and decrease in bile salts were also observed in all exposed groups of males. Urine protein 

and glucose concentrations were increased in all exposed male groups. Urinary aspartate 

aminotransferase and N-acetyl-β-D-glucosaminidase activities were significantly increased in all 

exposed groups of both sexes. Estrous cycles were longer in females at ≥ 15,000 ppm.

All animals in all exposed groups of male and female rats showed liver hypertrophy, eosinophilic 

hyaline droplets in the kidney, and congestion and pigmentation of the spleen. Hematopoietic 

cell proliferation of the spleen was seen in all males of all exposed groups and in all females of 

all exposed groups except for one female at 3750 ppm. None of the controls animals showed 

these lesions of liver, kidney, or spleen. Bone marrow hyperplasia was seen in all exposed 

female groups and in male groups at ≥ 3750 ppm. The incidence of nephropathy was increased 

in females at 15,000 and 30,000 ppm. Nephropathy was observed in all groups of males (control 

and exposed), with increased severity in the exposed groups. All exposed males had increased 

concentrations of α-2u-globulin in the kidneys. Thyroid gland follicular cell hypertrophy 

occurred in all males and females at ≥ 3750 ppm. Incidences of inflammation and transitional 

cell hyperplasia in the urinary bladder were increased in females at 30,000 ppm.
Draft HPV1/HPV2 Chemical Data Adequacy Review (May 2013) CASRN 84-65-1

57

CONCLUSIONS

Under the conditions of this study, the 14-week LOAEL in rats was 1875 ppm (approximately 

135 mg/kg-bw/day). A NOAEL was not established.

STUDY RELIABILITY

[1] Reliable without restrictions.

REFERENCES

NTP (National Toxicology Program). 2005. Toxicology and carcinogenesis studies of 

anthraquinone (CAS No. 84-65-1) in F344/N rats and B6C3F1 mice (feed studies). NTP TR494. September, 2005. Text in quotes is taken verbatim from NTP (2005).
Draft HPV1/HPV2 Chemical Data Adequacy Review (May 2013) CASRN 84-65-1

58

Study 2: 14-Week repeated-dose oral toxicity study in mice (NTP, 2005)

Title: Toxicology and carcinogenesis studies of anthraquinone (CAS No. 84-65-1) in F344/N

rats and B6C3F1 mice (feed studies) 

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: 9,10-Anthracenedione, CASRN 84-65-1

Remarks: 99.8% pure

METHOD

Method/guideline followed: NTP Protocols (Experimental design presented in Table 1 of NTP 

[2005]).

Test type: 14-Week Oral Toxicity Study in Mice 

GLP compliant?: Yes

Year study performed: 1994

Test species: B6C3F1 mice (male and female)

Number of animals/sex/dose: 10

Route of administration: Oral – dietary

Vehicle: None

Exposure duration: 14 weeks

Doses/concentrations tested: 0, 1875, 3750, 7500, 15,000, and 30,000 ppm (approximately 

equivalent to 250, 500, 1050, 2150, and 4300 mg/kg-bw/day, respectively, for males, and 300, 

640, 1260, 2600, and 5300 mg/kg-bw/day, respectively, for females) 

Statistical methods used: “Survival Analyses: The probability of survival was estimated by the 

product-limit procedure of Kaplan and Meier (1958) and is presented in the form of graphs. 

Animals found dead of other than natural causes or missing were censored from the survival 

analyses; animals dying from natural causes were not censored. Statistical analyses for possible 

dose-related effects on survival used Cox’s (1972) method for testing two groups for equality 

and Tarone’s (1975) life table test to identify dose-related trends. All reported P values for the 

survival analyses are two sided.”
Draft HPV1/HPV2 Chemical Data Adequacy Review (May 2013) CASRN 84-65-1

59

“Calculation of Incidence: The incidences of neoplasms or nonneoplastic lesions are 

presented... as the numbers of animals bearing such lesions at a specific anatomic site and the 

numbers of animals with that site examined microscopically. For calculation of statistical 

significance, the incidences of most neoplasms... and all nonneoplastic lesions are given as the 

numbers of animals affected at each site examined microscopically. However, when 

macroscopic examination was required to detect neoplasms in certain tissues (e.g., harderian 

gland, intestine, mammary gland, and skin) before microscopic evaluation, or when neoplasms 

had multiple potential sites of occurrence (e.g., leukemia or lymphoma), the denominators 

consist of the number of animals on which a necropsy was performed.” Survival-adjusted 

neoplasm rates for each group and each site-specific neoplasm are also presented. The “survivaladjusted rate (based on the Poly-3 method described below) accounts for differential mortality by 

assigning a reduced risk of neoplasm, proportional to the third power of the fraction of time on 

study, to animals that do not reach terminal sacrifice.”

“Analysis of Neoplasm and Nonneoplastic Lesion Incidences: The Poly-k test (Bailer and 

Portier, 1988; Portier and Bailer, 1989; Piegorsch and Bailer, 1997) was used to assess neoplasm 

and nonneoplastic lesion prevalence. This test is a survival-adjusted quantal-response procedure 

that modifies the Cochran-Armitage linear trend test to take survival differences into account. 

More specifically, this method modifies the denominator in the quantal estimate of lesion 

incidence to approximate more closely the total number of animal years at risk. For analysis of a 

given site, each animal is assigned a risk weight. This value is one if the animal had a lesion at 

that site or if it survived until terminal sacrifice; if the animal died prior to terminal sacrifice and 

did not have a lesion at that site, its risk weight is the fraction of the entire study time that it 

survived, raised to the kth power. This method yields a lesion prevalence rate that depends only 

upon the choice of a shape parameter for a Weibull hazard function describing cumulative lesion 

incidence over time (Bailer and Portier, 1988). Unless otherwise specified, a value of k=3 was 

used in the analysis of site-specific lesions. This value was recommended by Bailer and Portier 

(1988) following an evaluation of neoplasm onset time distributions for a variety of site-specific 

neoplasms in control F344 rats and B6C3F1 mice (Portier et al., 1986). Bailer and Portier 

(1988) showed that the Poly-3 test gave valid results if the true value of k was anywhere in the 

range from 1 to 5. A further advantage of the Poly-3 method is that it does not require lesion 

lethality assumptions. Variation introduced by the use of risk weights, which reflect differential 

mortality, was accommodated by adjusting the variance of the Poly-3 statistic as recommended

by Bieler and Williams (1993). Tests of significance included pairwise comparisons of each 

exposed group with controls and a test for an overall exposure-related trend. Continuitycorrected Poly-3 tests were used in the analysis of lesion incidence, and reported P values are 

one sided... For neoplasms and nonneoplastic lesions detected at the interim evaluations, the 

Fisher exact test (Gart et al., 1979), a procedure based on the overall proportion of affected 

animals, was used.”

“Analysis of Continuous Variables: Two approaches were employed to assess the significance

of pairwise comparisons between exposed and control groups in the analysis of continuous 

variables. Organ and body weight data, which historically have approximately normal 

distributions, were analyzed with the parametric multiple comparison procedures of Dunnett 

(1955) and Williams (1971, 1972). Soluble protein and α-2u-globulin concentrations were 

analyzed by Dunnett’s one-tailed t-test for comparing means to the 0 ppm group and Duncan’s 
Draft HPV1/HPV2 Chemical Data Adequacy Review (May 2013) CASRN 84-65-1

60

multiple range test for comparing all means to each other. Hematology, clinical chemistry, 

urinalysis, toxicokinetic, spermatid, and epididymal spermatozoal data, which have typically

skewed distributions, were analyzed using the nonparametric multiple comparison methods of 

Shirley (1977) (as modified by Williams, 1986) and Dunn (1964). Jonckheere’s test 

(Jonckheere, 1954) was used to assess the significance of the dose-related trends and to 

determine whether a trend-sensitive test (Williams’ or Shirley’s test) was more appropriate for 

pairwise comparisons than a test that does not assume a monotonic dose-related trend (Dunnett’s 

or Dunn’s test). Prior to statistical analysis, extreme values identified by the outlier test of Dixon 

and Massey (1957) were examined by NTP personnel, and implausible values were eliminated 

from the analysis. Average severity values were analyzed for significance with the MannWhitney U test (Hollander and Wolfe, 1973). Because vaginal cytology data are proportions (the 

proportion of the observation period that an animal was in a given estrous stage), an arcsine 

transformation was used to bring the data into closer conformance with a normality assumption. 

Treatment effects were investigated by applying a multivariate analysis of variance (Morrison, 

1976) to the transformed data to test for simultaneous equality of measurements across exposure

concentrations.”

Test method/conditions remarks: “Male and female... B6C3F1 mice were obtained from 

Taconic Farms (Germantown, NY). On receipt,... mice were approximately 4 weeks old... 

[M]ice were quarantined for 13 (males) or 14 (females) days;... mice were approximately

6 to 7 weeks old on the first day of the studies. Before initiation of the studies, five male and 

five female... mice were randomly selected for parasite evaluation and gross observation for 

evidence of disease. Serologic analyses were performed on up to five male and five female 

sentinel... mice 1 month after study start and on up to five male and five female control... 

sentinel mice at study termination using the protocols of the NTP Sentinel Animal Program...”

Groups of 10 male and 10 female mice were administered diets containing 0, 1875, 3750, 7500, 

15,000, or 30,000 ppm (approximately equivalent to 250, 500, 1050, 2150, and 4300 mg/kgbw/day, respectively, for males, and 300, 640, 1260, 2600, and 5300 mg/kg-bw/day, 

respectively, for females) for 14 weeks. “Water was available ad libitum and feed was available 

ad libitum... [F]emale mice were housed five per cage... and male mice were housed 

individually. Clinical findings were recorded and animals were weighed weekly and at the end 

of the studies. Feed consumption was recorded twice weekly or once weekly (male mice).”

Blood was collected from the retroorbital sinus of all core study mice surviving to the end of the 

studies for hematology analyses. “Blood samples for hematology analyses were placed into 

microcollection tubes containing potassium EDTA. Erythrocyte, platelet, and leukocyte counts, 

hematocrit values, hemoglobin concentration, mean cell volume, mean cell hemoglobin, and 

mean cell hemoglobin concentration were determined using a Serono-Baker System 9000 

hematology analyzer (Serono-Baker Diagnostics, Allentown, PA). Differential leukocyte counts 

and erythrocyte and platelet morphologies were determined microscopically from blood smears 

stained with Wright-Giemsa stain on a Hema-Tek slide stainer (Miles Laboratory, Ames 

Division, Elkhart, IN). A Miller disc was used to determine reticulocyte counts from smears 

prepared with blood stained with new methylene blue.” The following hematology parameters 

were measured: erythrocyte, reticulocyte, and platelet counts; hematocrit values; hemoglobin 
Draft HPV1/HPV2 Chemical Data Adequacy Review (May 2013) CASRN 84-65-1

61

concentration; erythrocyte and platelet morphology; mean cell volume; mean cell hemoglobin; 

mean cell hemoglobin concentration; and leukocyte count and differentials. 

“At the end of the 14-week studies, samples were collected for sperm motility evaluations on...

mice exposed to 0, 7500, 15,000, and 30,000 ppm.” “The following parameters were evaluated: 

spermatid heads per testis and per gram testis, spermatid counts, and epididymal spermatozoal 

motility and concentration. The left cauda epididymis, left epididymis, and left testis were 

weighed. Vaginal samples were collected for up to 12 consecutive days prior to the end of the 

studies from... 0, 7500, 15,000, and 30,000 ppm female... mice for vaginal cytology 

evaluations. The following parameters were evaluated: estrous cycle length and relative 

frequency of the estrous stages.” 

Necropsies were performed on all animals. “The heart, right kidney, liver, lungs, right testis, and

thymus were weighed. Tissues for microscopic examination were fixed and preserved in 10% 

neutral buffered formalin, processed and trimmed, embedded in paraffin, sectioned to a thickness 

of 4 to 6 μm, and stained with hematoxylin and eosin. Complete histopathologic examinations 

were performed on all core study 0 and 30,000 ppm mice.” “In addition to gross lesions and 

tissue masses, the following tissues [and organs] were examined microscopically: adrenal gland, 

bone with marrow, brain, clitoral gland, esophagus, gallbladder..., heart and aorta, large intestine 

(cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, liver, lung and

mainstem bronchi, lymph nodes (mandibular and mesenteric), mammary gland (with adjacent 

skin), nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland,

salivary gland, spleen, stomach (forestomach and glandular), testis (with epididymis and seminal 

vesicle), thymus, thyroid gland, trachea, urinary bladder, and uterus. In addition,... the liver and 

urinary bladder of mice were examined in all lower exposure groups.”

Endpoint value(s): LOAEL = 1875 ppm (250 and 300 mg/kg-bw/day in males and females, 

respectively) based on increased liver weight in both sexes, responsive anemia in females, 

cytoplasmic alteration of the urinary bladder with concentration-related increases in severity in 

both sexes, and increased incidences of hematopoietic cell proliferation and pigmentation of the 

spleen in males. A NOAEL was not established.

Results remarks: All mice survived to scheduled termination. Mean body weight and food 

consumption were similar between the treated and control groups. For all exposed groups, 

absolute and relative mean liver weights were greater than in controls. A responsive anemia was 

present in females of all exposed groups and in males at ≥ 7500 ppm by week 14. “The anemia 

was demonstrated by decreased hematocrit values, hemoglobin concentrations, and/or 

erythrocyte counts in males exposed to 7500 ppm or greater and in all groups of exposed 

females. An erythropoietic response to the anemia was demonstrated by increased reticulocyte 

counts in 15,000 and 30,000 ppm males and in [all] exposed groups of females. Accompanying 

increased reticulocyte counts were minimal increases in mean cell volumes and mean cell 

hemoglobin values. Mean cell hemoglobin concentrations were minimally increased in 15,000 

and 30,000 ppm males and females. There were minimal to mild exposure concentration-related 

increases in platelet counts in 15,000 and 30,000 ppm males and all exposed groups of females.”
Draft HPV1/HPV2 Chemical Data Adequacy Review (May 2013) CASRN 84-65-1

62

At ≥ 3750 ppm, the incidences and severity of centrilobular hypertrophy of the liver were 

significantly greater than in controls. Increased incidences of hematopoietic cell proliferation 

and pigmentation of the spleen occurred in all exposed male groups compared with 

controls. Cytoplasmic alteration of the transitional epithelium of the urinary bladder was 

observed in all exposed mice with concentration-related increases in severity.

CONCLUSIONS

Under the conditions of this study, the 14-week LOAEL in mice was 1875 ppm (250 and 

300 mg/kg-bw/day in males and females, respectively). A NOAEL was not established.

STUDY RELIABILITY

[2] Reliable with restrictions. Clinical chemistry and urinalysis parameters were not measured.

REFERENCES

NTP (National Toxicology Program). 2005. Toxicology and carcinogenesis studies of 

anthraquinone (CAS No. 84-65-1) in F344/N rats and B6C3F1 mice (feed studies). NTP TR494. September, 2005. Text in quotes is taken verbatim from NTP (2005).
Draft HPV1/HPV2 Chemical Data Adequacy Review (May 2013) CASRN 84-65-1

63

Study 3: 105-Week repeated-dose oral toxicity study in rats (NTP, 2005)

Title: Toxicology and carcinogenesis studies of anthraquinone (CAS No. 84-65-1) in F344/N 

rats and B6C3F1 mice (feed studies)

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: 9,10-Anthracenedione, CASRN 84-65-1

Remarks: 99.8% pure

METHOD

Method/guideline followed: NTP Protocols (Experimental design presented in Table 1 of NTP 

[2005]).

Test type: 105-Week Oral Toxicity Study in Rats

GLP compliant?: Yes

Year study performed: 1996

Test species: Fischer 344 rats (male and female)

Number of animals/sex/dose: 60 at 0 and 3750 ppm; 50 at 469, 938, and 1875 ppm

Route of administration: Oral – dietary

Vehicle: None

Exposure duration: 105 weeks

Doses/concentrations tested: 0, 469, 938, 1875, or 3750 ppm (approximately equivalent to 0, 

20, 45, 90, or 180 mg/kg-bw/day, respectively, for males and 0, 25, 50, 100, or 200 mg/kgbw/day, respectively, for females)

Statistical methods used: “Survival Analyses: The probability of survival was estimated by the 

product-limit procedure of Kaplan and Meier (1958) and is presented in the form of graphs. 

Animals found dead of other than natural causes or missing were censored from the survival 

analyses; animals dying from natural causes were not censored. Statistical analyses for possible 

dose-related effects on survival used Cox’s (1972) method for testing two groups for equality 

and Tarone’s (1975) life table test to identify dose-related trends. All reported P values for the 

survival analyses are two sided.”
Draft HPV1/HPV2 Chemical Data Adequacy Review (May 2013) CASRN 84-65-1

64

“Calculation of Incidence: The incidences of neoplasms or nonneoplastic lesions are 

presented... as the numbers of animals bearing such lesions at a specific anatomic site and the 

numbers of animals with that site examined microscopically. For calculation of statistical 

significance, the incidences of most neoplasms... and all nonneoplastic lesions are given as the 

numbers of animals affected at each site examined microscopically. However, when 

macroscopic examination was required to detect neoplasms in certain tissues (e.g., harderian 

gland, intestine, mammary gland, and skin) before microscopic evaluation, or when neoplasms 

had multiple potential sites of occurrence (e.g., leukemia or lymphoma), the denominators 

consist of the number of animals on which a necropsy was performed.” Survival-adjusted 

neoplasm rates for each group and each site-specific neoplasm are also presented. The “survivaladjusted rate (based on the Poly-3 method described below) accounts for differential mortality by 

assigning a reduced risk of neoplasm, proportional to the third power of the fraction of time on 

study, to animals that do not reach terminal sacrifice.”

“Analysis of Neoplasm and Nonneoplastic Lesion Incidences: The Poly-k test (Bailer and 

Portier, 1988; Portier and Bailer, 1989; Piegorsch and Bailer, 1997) was used to assess neoplasm 

and nonneoplastic lesion prevalence. This test is a survival-adjusted quantal-response procedure 

that modifies the Cochran-Armitage linear trend test to take survival differences into account. 

More specifically, this method modifies the denominator in the quantal estimate of lesion 

incidence to approximate more closely the total number of animal years at risk. For analysis of a 

given site, each animal is assigned a risk weight. This value is one if the animal had a lesion at 

that site or if it survived until terminal sacrifice; if the animal died prior to terminal sacrifice and 

did not have a lesion at that site, its risk weight is the fraction of the entire study time that it 

survived, raised to the kth power. This method yields a lesion prevalence rate that depends only 

upon the choice of a shape parameter for a Weibull hazard function describing cumulative lesion 

incidence over time (Bailer and Portier, 1988). Unless otherwise specified, a value of k=3 was 

used in the analysis of site-specific lesions. This value was recommended by Bailer and Portier 

(1988) following an evaluation of neoplasm onset time distributions for a variety of site-specific 

neoplasms in control F344 rats and B6C3F1 mice (Portier et al., 1986). Bailer and Portier 

(1988) showed that the Poly-3 test gave valid results if the true value of k was anywhere in the 

range from 1 to 5. A further advantage of the Poly-3 method is that it does not require lesion 

lethality assumptions. Variation introduced by the use of risk weights, which reflect differential 

mortality, was accommodated by adjusting the variance of the Poly-3 statistic as recommended

by Bieler and Williams (1993). Tests of significance included pairwise comparisons of each 

exposed group with controls and a test for an overall exposure-related trend. Continuitycorrected Poly-3 tests were used in the analysis of lesion incidence, and reported P values are 

one sided... For neoplasms and nonneoplastic lesions detected at the interim evaluations, the 

Fisher exact test (Gart et al., 1979), a procedure based on the overall proportion of affected 

animals, was used.”

“Analysis of Continuous Variables: Two approaches were employed to assess the significance

of pairwise comparisons between exposed and control groups in the analysis of continuous 

variables. Organ and body weight data, which historically have approximately normal 

distributions, were analyzed with the parametric multiple comparison procedures of Dunnett 

(1955) and Williams (1971, 1972). Soluble protein and α-2u-globulin concentrations were 

analyzed by Dunnett’s one-tailed t-test for comparing means to the 0 ppm group and Duncan’s 
Draft HPV1/HPV2 Chemical Data Adequacy Review (May 2013) CASRN 84-65-1

65

multiple range test for comparing all means to each other. Hematology, clinical chemistry, 

urinalysis, toxicokinetic, spermatid, and epididymal spermatozoal data, which have typically

skewed distributions, were analyzed using the nonparametric multiple comparison methods of 

Shirley (1977) (as modified by Williams, 1986) and Dunn (1964). Jonckheere’s test 

(Jonckheere, 1954) was used to assess the significance of the dose-related trends and to 

determine whether a trend-sensitive test (Williams’ or Shirley’s test) was more appropriate for 

pairwise comparisons than a test that does not assume a monotonic dose-related trend (Dunnett’s 

or Dunn’s test). Prior to statistical analysis, extreme values identified by the outlier test of Dixon 

and Massey (1957) were examined by NTP personnel, and implausible values were eliminated 

from the analysis. Average severity values were analyzed for significance with the MannWhitney U test (Hollander and Wolfe, 1973). Because vaginal cytology data are proportions (the 

proportion of the observation period that an animal was in a given estrous stage), an arcsine 

transformation was used to bring the data into closer conformance with a normality assumption. 

Treatment effects were investigated by applying a multivariate analysis of variance (Morrison, 

1976) to the transformed data to test for simultaneous equality of measurements across exposure

concentrations.”

Test method/conditions remarks: “Groups of 60 male and 60 female rats were fed diets

containing 0 or 3750 ppm anthraquinone, and 50 male and 50 female rats received 469, 938, or 

1875 ppm anthraquinone for 105 weeks.” Doses were approximately equal to 0, 20, 45, 90, or 

180 mg/kg-bw/day, respectively, for males and 0, 25, 50, 100, or 200 mg/kg-bw/day, 

respectively, for females. Five male and five female 0 and 3750 ppm rats were evaluated at 3 

months for histopathology and α-2u-globulin concentrations and at 12 months for organ weights 

and histopathology. “Additional groups of 18 male rats given 469, 938, 1875, or 3750 ppm for 

8 days and 10 male and 10 female rats given 469, 938, or 1875 ppm for 3, 6, 12, or 18 months 

were designated for toxicokinetic studies. Toxicokinetic studies were also conducted on

10 male and 10 female randomly selected 3750 ppm core study rats at 3, 6, 12, and 18 months.”

“Male and female F344/N rats were obtained from Taconic Laboratory Animals and Services

(Germantown, NY) for use in the 2-year studies. Rats... were quarantined for 11 (males) or

12 (females) days before the beginning of the studies. Five male and five female rats... were 

randomly selected for parasite evaluation and gross observation of disease. Rats... were 

approximately 6 to 7 weeks old at the beginning of the studies. The health of the animals was 

monitored during the studies according to the protocols of the NTP Sentinel Animal Program.”

“Rats were housed three (males) or five (females) per cage... Feed and water were available

ad libitum. Feed consumption was measured every 4 weeks. Cages were changed twice weekly; 

racks were changed and rotated every 2 weeks... All animals were observed twice daily.

Clinical findings were recorded every 4 weeks and at the end of the studies. Body weights were 

recorded at the beginning of the studies, on day 8, every 4 weeks, and at the end of the 

studies...” 

“Complete necropsies and microscopic examinations were performed on core study rats...

Interim evaluations of 0 and 3750 ppm rats were conducted at 3 and 12 months; left and right 

kidneys and the liver were weighed at 12 months. At necropsy, all organs and tissues were 

examined for grossly visible lesions, and all major tissues were fixed and preserved in 10%
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neutral buffered formalin, processed and trimmed, embedded in paraffin, sectioned to a thickness 

of 5 to 6 μm, and stained with hematoxylin and eosin for microscopic examination. For all 

paired organs (e.g., adrenal gland, kidney, ovary), samples from each organ were examined.”

“In addition to gross lesions and tissue masses, the following tissues and organs were examined 

microscopically: adrenal gland, bone with marrow, brain, clitoral gland, esophagus,... heart and 

aorta, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), 

kidney, liver, lung and mainstem bronchi, lymph nodes (mandibular and mesenteric), mammary 

gland (with adjacent skin)..., nose, ovary, pancreas, pancreatic islets, parathyroid gland, pituitary 

gland, preputial gland, prostate gland, salivary gland, spleen, stomach (forestomach and 

glandular), testis (with epididymis and seminal vesicle), thymus, thyroid gland, trachea, urinary 

bladder, and uterus.” 

“Microscopic evaluations were completed by the study laboratory pathologist, and the pathology 

data were entered into the Toxicology Data Management System. The slides, paraffin blocks, 

and residual wet tissues were sent to the NTP Archives for inventory, slide/block match, and wet 

tissue audit. The slides, individual animal data records, and pathology tables were evaluated by 

an independent quality assessment laboratory. The individual animal records and tables were 

compared for accuracy, the slide and tissue counts were verified, and the histotechnique was 

evaluated. For the 2-year studies, a quality assessment pathologist evaluated slides from all 

tumors and all potential target organs, which included the bone marrow, kidney, liver, spleen, 

thyroid gland, and urinary bladder of male and female rats... The quality assessment report and 

the reviewed slides were submitted to the NTP Pathology Working Group (PWG) chairperson, 

who reviewed the selected tissues and addressed any inconsistencies in the diagnoses made by 

the laboratory and quality assessment pathologists. Representative histopathology slides 

containing examples of lesions related to chemical administration, examples of disagreements in 

diagnoses between the laboratory and quality assessment pathologists, or lesions of general 

interest were presented by the chairperson to the PWG for review. The PWG consisted of the 

quality assessment pathologist and other pathologists experienced in rodent toxicologic 

pathology. This group examined the tissues without any knowledge of dose groups or previously 

rendered diagnoses. When the PWG consensus differed from the opinion of the laboratory 

pathologist, the diagnosis was changed. Final diagnoses for reviewed lesions represent a 

consensus between the laboratory pathologist, reviewing pathologist(s), and the PWG. Details of

these review procedures have been described, in part, by Maronpot and Boorman (1982) and 

Boorman et al. (1985). For subsequent analyses of the pathology data, the decision of whether to 

evaluate the diagnosed lesions for each tissue type separately or combined was generally based 

on the guidelines of McConnell et al. (1986).”

Endpoint value(s): LOAEL (systemic toxicity) = 469 ppm (approximately 20 and 25 mg/kgbw/day in males and females, respectively) based on kidney, spleen, liver, and bone marrow 

effects and > 10% reduction in terminal body weight in females. A NOAEL was not established.

Increased incidences of neoplasms were seen in kidney, urinary bladder, and liver at 938 ppm 

(approximately 45 and 50 mg/kg-bw/day in males and females, respectively) and higher. 

Incidences of mononuclear cell leukemia were less in exposed groups than in controls.
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Results remarks: Survival was not affected by treatment. Food consumption was similar 

between the treated and control groups. Body weight was reduced for exposed males during the 

latter part of the study and for exposed females throughout most of the study. At the end of the 

exposure, body weights of males were 94, 93, 96 and 93% of control values and body weights of 

females were 89, 85, 83 and 79% of control values at 469, 938, 1875 and 3750 ppm, 

respectively; no statistical analysis of body weights was provided. 

Non-neoplastic Effects at 2 Years: 

Increased incidences of hyaline droplet accumulation were seen in the kidneys of all exposed 

groups of males and females except at 938 ppm in males. Other kidney lesions seen at increased 

incidences at all doses included mineralization of the medulla in males, hyperplasia of the 

transitional epithelium in males, nephropathy and pigmentation in females, and renal tubular 

hyperplasia in females. Severities of nephropathy were increased in males relative to control 

rats. Incidences of kidney pigmentation were increased in males at 938 ppm and higher. 

Incidences of mineralization of the medulla were increased in females at 938 and 1875 ppm. 

Increased incidences of hyperplasia of the transitional epithelium of the renal pelvis were seen in 

females at 469, 938, and 3750 ppm. The concentration of α-2u-globulin in kidney homogenates 

at 3 months was greater in 3750 ppm males (only dose and time examined) but lower in 3750 

ppm females than in homogenates from the respective control groups. 

Liver lesions seen at increased incidences at all doses included eosiniphilic foci, cystic 

degeneration, and inflammation in both sexes; cytoplasmic vacuolation in males; and angiectasis, 

centrilobular hypertrophy and mixed cell foci in females. Incidences of basophilic foci were 

increased in males at 469 and 938 ppm and in females at 469 and 3750 ppm. In males, 

incidences of mixed cell foci were elevated at 469, 938, and 1875 ppm and incidences of 

centrilobular hypertrophy were elevated at 938, 1875, and 3750 ppm. 

Incidences of congestion, pigmentation, and hematopoietic cell proliferation of the spleen were

greater in all groups of exposed males and females than in the controls. Incidences of bone 

marrow hyperplasia were increased in males at doses of 938 ppm or higher and in females at 469 

ppm, and the incidences of bone marrow atrophy were increased in all groups of exposed 

females. 

Neoplastic Effects at 2 Years:

“The incidences of renal tubule adenoma and renal tubule adenoma or carcinoma (combined) 

occurred with positive trends and were increased in all exposed groups of female rats. The 

incidences of renal tubule adenoma in all exposed groups of male rats exceeded the historical 

control range, and the incidence was significantly increased in the 938 ppm group.” “The 

incidence of urinary bladder transitional epithelial papilloma was significantly greater in 

1875 ppm males than in the control group, and the incidences in groups of males exposed to 938 

ppm or greater exceeded the historical control range. There were positive trends in the 

incidences of transitional epithelial hyperplasia and papilloma or carcinoma (combined) of the 

urinary bladder in females.” “The incidences of hepatocellular adenoma or carcinoma

(combined) were slightly increased in exposed males and females; the incidences in groups of 

females exposed to 938 ppm or greater exceeded the historical control range.” “The incidences 
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of mononuclear cell leukemia were significantly less in all exposed groups than in the controls at 

2 years, and the incidences were less than the historical control ranges.”

CONCLUSIONS

NTP concluded that there was some evidence of carcinogenic activity of anthraquinone in male 

rats and clear evidence of carcinogenic activity in female rats. The 105-week LOAEL for 

systemic toxicity was 469 ppm in the diet (approximately 20 and 25 mg/kg-bw/day in males and 

females, respectively); a NOAEL was not established.

STUDY RELIABILITY

[1] Reliable without restrictions. 

REFERENCES

NTP (National Toxicology Program). 2005. Toxicology and carcinogenesis studies of 

anthraquinone (CAS No. 84-65-1) in F344/N rats and B6C3F1 mice (feed studies). NTP TR494. September, 2005. Text in quotes is taken verbatim from NTP (2005).
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Study 4: 105-Week repeated-dose oral toxicity study in mice (NTP, 2005)

Title: Toxicology and carcinogenesis studies of anthraquinone (CAS No. 84-65-1) in F344/N 

rats and B6C3F1 mice (feed studies)

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: 9,10-Anthracenedione, CASRN 84-65-1

Remarks: 99.8% pure

METHOD

Method/guideline followed: NTP Protocols (Experimental design presented in Table 1 of NTP 

[2005]).

Test type: 105-Week Oral Toxicity Study in Mice

GLP compliant?: Yes

Year study performed: 1996

Test species: B6C3F1 mice (male and female)

Number of animals/sex/dose: 50

Route of administration: Oral – dietary

Vehicle: None

Exposure duration: 105 weeks

Doses/concentrations tested: 0, 833, 2500, and 7500 ppm (approximately equivalent to 90, 

265, and 825 mg/kg-bw/day, respectively, for males and 80, 235, and 745 mg/kg-bw/day, 

respectively, for females)

Statistical methods used: “Survival Analyses: The probability of survival was estimated by the 

product-limit procedure of Kaplan and Meier (1958) and is presented in the form of graphs. 

Animals found dead of other than natural causes or missing were censored from the survival 

analyses; animals dying from natural causes were not censored. Statistical analyses for possible 

dose-related effects on survival used Cox’s (1972) method for testing two groups for equality 

and Tarone’s (1975) life table test to identify dose-related trends. All reported P values for the 

survival analyses are two sided.”
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“Calculation of Incidence: The incidences of neoplasms or nonneoplastic lesions are 

presented... as the numbers of animals bearing such lesions at a specific anatomic site and the 

numbers of animals with that site examined microscopically. For calculation of statistical 

significance, the incidences of most neoplasms... and all nonneoplastic lesions are given as the 

numbers of animals affected at each site examined microscopically. However, when 

macroscopic examination was required to detect neoplasms in certain tissues (e.g., harderian 

gland, intestine, mammary gland, and skin) before microscopic evaluation, or when neoplasms 

had multiple potential sites of occurrence (e.g., leukemia or lymphoma), the denominators 

consist of the number of animals on which a necropsy was performed.” Survival-adjusted 

neoplasm rates for each group and each site-specific neoplasm are also presented. The “survivaladjusted rate (based on the Poly-3 method described below) accounts for differential mortality by 

assigning a reduced risk of neoplasm, proportional to the third power of the fraction of time on 

study, to animals that do not reach terminal sacrifice.”

“Analysis of Neoplasm and Nonneoplastic Lesion Incidences: The Poly-k test (Bailer and 

Portier, 1988; Portier and Bailer, 1989; Piegorsch and Bailer, 1997) was used to assess neoplasm 

and nonneoplastic lesion prevalence. This test is a survival-adjusted quantal-response procedure 

that modifies the Cochran-Armitage linear trend test to take survival differences into account. 

More specifically, this method modifies the denominator in the quantal estimate of lesion 

incidence to approximate more closely the total number of animal years at risk. For analysis of a 

given site, each animal is assigned a risk weight. This value is one if the animal had a lesion at 

that site or if it survived until terminal sacrifice; if the animal died prior to terminal sacrifice and 

did not have a lesion at that site, its risk weight is the fraction of the entire study time that it 

survived, raised to the kth power. This method yields a lesion prevalence rate that depends only 

upon the choice of a shape parameter for a Weibull hazard function describing cumulative lesion 

incidence over time (Bailer and Portier, 1988). Unless otherwise specified, a value of k=3 was 

used in the analysis of site-specific lesions. This value was recommended by Bailer and Portier 

(1988) following an evaluation of neoplasm onset time distributions for a variety of site-specific 

neoplasms in control F344 rats and B6C3F1 mice (Portier et al., 1986). Bailer and Portier 

(1988) showed that the Poly-3 test gave valid results if the true value of k was anywhere in the 

range from 1 to 5. A further advantage of the Poly-3 method is that it does not require lesion 

lethality assumptions. Variation introduced by the use of risk weights, which reflect differential 

mortality, was accommodated by adjusting the variance of the Poly-3 statistic as recommended

by Bieler and Williams (1993). Tests of significance included pairwise comparisons of each 

exposed group with controls and a test for an overall exposure-related trend. Continuitycorrected Poly-3 tests were used in the analysis of lesion incidence, and reported P values are 

one sided... For neoplasms and nonneoplastic lesions detected at the interim evaluations, the 

Fisher exact test (Gart et al., 1979), a procedure based on the overall proportion of affected 

animals, was used.”

“Analysis of Continuous Variables: Two approaches were employed to assess the significance

of pairwise comparisons between exposed and control groups in the analysis of continuous 

variables. Organ and body weight data, which historically have approximately normal 

distributions, were analyzed with the parametric multiple comparison procedures of Dunnett 

(1955) and Williams (1971, 1972). Soluble protein and α-2u-globulin concentrations were 

analyzed by Dunnett’s one-tailed t-test for comparing means to the 0 ppm group and Duncan’s 
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multiple range test for comparing all means to each other. Hematology, clinical chemistry, 

urinalysis, toxicokinetic, spermatid, and epididymal spermatozoal data, which have typically

skewed distributions, were analyzed using the nonparametric multiple comparison methods of 

Shirley (1977) (as modified by Williams, 1986) and Dunn (1964). Jonckheere’s test 

(Jonckheere, 1954) was used to assess the significance of the dose-related trends and to 

determine whether a trend-sensitive test (Williams’ or Shirley’s test) was more appropriate for 

pairwise comparisons than a test that does not assume a monotonic dose-related trend (Dunnett’s 

or Dunn’s test). Prior to statistical analysis, extreme values identified by the outlier test of Dixon 

and Massey (1957) were examined by NTP personnel, and implausible values were eliminated 

from the analysis. Average severity values were analyzed for significance with the MannWhitney U test (Hollander and Wolfe, 1973). Because vaginal cytology data are proportions (the 

proportion of the observation period that an animal was in a given estrous stage), an arcsine 

transformation was used to bring the data into closer conformance with a normality assumption. 

Treatment effects were investigated by applying a multivariate analysis of variance (Morrison, 

1976) to the transformed data to test for simultaneous equality of measurements across exposure

concentrations.”

Test method/conditions remarks: Groups of 50 male and 50 female mice were fed diets 

containing 0, 833, 2500, or 7500 ppm anthraquinone (approximately equivalent to 90, 265, and 

825 mg/kg-bw/day, respectively, for males and 80, 235, and 745 mg/kg-bw/day, respectively, for 

females) for 105 weeks. “Additional groups of 36 male mice given 833, 2500, or 7500 ppm for 

8 days and 10 male and 10 female mice given 833, 2500, or 7500 ppm for 12 months were 

designated for toxicokinetic studies.” 

“Male and female... B63CF1 mice were obtained from Taconic Laboratory Animals and 

Services (Germantown, NY) for use in the 2-year studies... [M]ice were quarantined for 

11 (males) or 12 (females) days before the beginning of the studies. Five male and five female...

mice were randomly selected for parasite evaluation and gross observation of disease... [M]ice 

were approximately 6 to 7 weeks old at the beginning of the studies. The health of the animals 

was monitored during the studies according to the protocols of the NTP Sentinel Animal

Program... [M]ice were housed one (males) or five (females) per cage. Feed and water were 

available ad libitum. Feed consumption was measured every 4 weeks. Cages were changed 

twice weekly or once weekly (male mice); racks were changed and rotated every 2 weeks.” 

“All animals were observed twice daily. Clinical findings were recorded every 4 weeks and at 

the end of the studies. Body weights were recorded at the beginning of the studies, on day 8, 

every 4 weeks, and at the end of the studies.” “Complete necropsies and microscopic 

examinations were performed on core study... mice... At necropsy, all organs and tissues were 

examined for grossly visible lesions, and all major tissues were fixed and preserved in 10%

neutral buffered formalin, processed and trimmed, embedded in paraffin, sectioned to a thickness 

of 5 to 6 μm, and stained with hematoxylin and eosin for microscopic examination. For all 

paired organs (e.g., adrenal gland, kidney, ovary), samples from each organ were examined. For 

extended evaluation of renal tubule proliferative lesions in male mice, kidneys were stepsectioned at 1-mm intervals, and additional sections were obtained from each kidney.”
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“In addition to gross lesions and tissue masses, the following tissues and organs were examined 

[microscopically]: adrenal gland, bone with marrow, brain, clitoral gland, esophagus, 

gallbladder..., heart and aorta, large intestine (cecum, colon, rectum), small intestine (duodenum,

jejunum, ileum), kidney, liver, lung and mainstem bronchi, lymph nodes (mandibular and 

mesenteric), mammary gland (with adjacent skin) (except male mice), nose, ovary, pancreas, 

pancreatic islets, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary 

gland, spleen, stomach (forestomach and glandular), testis (with epididymis and seminal vesicle), 

thymus, thyroid gland, trachea, urinary bladder, and uterus.” 

“Microscopic evaluations were completed by the study laboratory pathologist, and the pathology 

data were entered into the Toxicology Data Management System. The slides, paraffin blocks, 

and residual wet tissues were sent to the NTP Archives for inventory, slide/block match, and wet 

tissue audit. The slides, individual animal data records, and pathology tables were evaluated by 

an independent quality assessment laboratory. The individual animal records and tables were 

compared for accuracy, the slide and tissue counts were verified, and the histotechnique was 

evaluated. For the 2-year studies, a quality assessment pathologist evaluated slides from all 

tumors and all potential target organs, which included the... liver, spleen, thyroid gland, and 

urinary bladder of male and female mice, pancreatic islets of male mice, and skin, stomach, and 

thymus of female mice. The quality assessment report and the reviewed slides were submitted to 

the NTP Pathology Working Group (PWG) chairperson, who reviewed the selected tissues and 

addressed any inconsistencies in the diagnoses made by the laboratory and quality assessment 

pathologists. Representative histopathology slides containing examples of lesions related to 

chemical administration, examples of disagreements in diagnoses between the laboratory and 

quality assessment pathologists, or lesions of general interest were presented by the chairperson 

to the PWG for review. The PWG consisted of the quality assessment pathologist and other

pathologists experienced in rodent toxicologic pathology. This group examined the tissues 

without any knowledge of dose groups or previously rendered diagnoses. When the PWG 

consensus differed from the opinion of the laboratory pathologist, the diagnosis was changed. 

Final diagnoses for reviewed lesions represent a consensus between the laboratory pathologist,

reviewing pathologist(s), and the PWG. Details of these review procedures have been described, 

in part, by Maronpot and Boorman (1982) and Boorman et al. (1985). For subsequent analyses 

of the pathology data, the decision of whether to evaluate the diagnosed lesions for each tissue 

type separately or combined was generally based on the guidelines of McConnell et al. (1986).”

Endpoint value(s): LOAEL (systemic toxicity) = 833 ppm (approximately 90 and 80 mg/kgbw/day in males and females, respectively) based on liver and urinary bladder effects. A 

NOAEL was not established. Increased incidences of neoplasms were seen in liver and thyroid.

Results remarks: Survival was decreased in males at 7500 ppm. Food consumption was 

similar between the treated and control groups. At 7500 ppm, body weight was reduced for 

males during the last 6 months of the study and for females at the end of the study.

Non-neoplastic Effects at 2 Years: 

Liver lesions seen at increased incidences at all doses included centrilobular hypertrophy in both 

sexes and hepatocyte erythrophagocytosis (hepatocyte cytoplasm distended with erythrocytes) in 

males. Increased incidences of fatty focal degeneration of hepatocytes were seen in males at 
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833 and 2500 ppm and in females at 7500 ppm. Increased incidences of eosinophilic liver foci 

were found in males at 2500 and 7500 ppm and in females at 833 and 7500 ppm. Increased 

incidences of liver hematopoietic cell proliferation and focal necrosis were seen only in males at 

7500 ppm. Incidences of intracytoplasmic inclusion body of the urinary bladder were increased 

in all exposed groups of males and females. Incidences of thyroid follicular cell hyperplasia in 

2500 and 7500 ppm males were greater than those in the controls. Incidences of hematopoietic 

cell proliferation of the spleen in males and females exposed to 7500 ppm were greater than 

those in the control groups. Pigmentation in the kidney was greater in 7500 ppm males than in 

the control. The incidence of pancreatic islet hyperplasia in 7500 ppm females was increased; 

however, incidences in 2500 and 7500 ppm males were decreased compared with control.

Neoplastic Effects at 2 Years:

“Incidences of hepatocellular neoplasms increased with a positive trend in male and female mice, 

and the incidences were increased in all exposed groups. The incidences of multiple 

hepatocellular adenomas were increased in all exposed groups of mice. Incidences of 

hepatoblastoma were... increased in males exposed to 2500 or 7500 ppm (incidences of multiple 

hepatoblastoma also increased), and one hepatoblastoma occurred in a 7500 ppm female. The 

incidences of liver neoplasms in exposed groups generally exceeded the historical control 

ranges... The incidences of thyroid follicular cell carcinoma and follicular cell adenoma or 

carcinoma (combined) in 7500 ppm females exceeded the historical control ranges...”

CONCLUSIONS

NTP concluded that there was clear evidence of carcinogenic activity of anthraquinone in male 

and female mice. Under the conditions of this study, the 105-week LOAEL in mice was 

833 ppm (approximately 90 and 80 mg/kg-bw/day in males and females, respectively). A 

NOAEL was not established.

STUDY RELIABILITY

[1] Reliable without restrictions. 

REFERENCES

NTP (National Toxicology Program). 2005. Toxicology and carcinogenesis studies of 

anthraquinone (CAS No. 84-65-1) in F344/N rats and B6C3F1 mice (feed studies). NTP TR494. September, 2005. Text in quotes is taken verbatim from NTP (2005).
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C-6. Genetic Toxicity – Gene Mutation

Summary of endpoint: Adequate 

Study 1: Mutagenicity study in Salmonella typhimurium (Butterworth et al., 2001)

Title: The preparation of anthraquinone used in the National Toxicology Program cancer 

bioassay was contaminated with the mutagen 9-nitroanthracene.

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: 9,10-Anthracenedione, CASRN 84-65-1 (commonly called anthraquinone and referred 

to as AQ in the study description) 

Remarks: “Commercial anthraquinone (AQ) is produced in large quantities by at least the 

following three production methods worldwide: oxidation of anthracene (AQ-OX), FriedelCrafts technology (AQ-FC) and Diels-Alder chemistry (AQ-DA), with the final product varying 

in color and purity. AQ-OX begins with anthracene produced from coal tar and different lots 

contain various contaminants, particularly the mutagenic isomers of nitroanthracene.” 

According to Butterworth et al. (2001), AQ produced by the AQ-FC and AQ-DA methods 

contain many fewer contaminants. “All AQ used commercially in the US is either AQ-FC or 

AQ-DA, rather than AQ-OX. Interestingly, the reagent grade material supplied to research 

laboratories is often AQ-OX.”

Four samples were tested, as follows: (1) A sample of the AQ-OX powder employed in the NTP 

2-year toxicology and carcinogenesis studies (NTP, 1999), designated NTP AQ-OX (Sample 

A07496 from Study 2); (2) NTP AQ-OX purified by 2X recrystalization from ethanol, 

designated purified NTP AQ-OX; (3) Technical grade AQ-FC typical of that in commercial use; 

and (4) Technical grade AQ-DA typical of that in commercial use. According to Butterworth et 

al. (2001), each of the four samples had a purity of 99% AQ based on high-performance liquid 

chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS) analysis 

conducted by DCV Group (Wilmington, DE) and Covance Laboratories (Leesburg, VA). 

Butterworth et al. (2001) reported that sample NTP AQ-OX contained 0.12% 9-nitroanthracene 

(9-NA) but the latter was not detected in any of the other samples at a limit of detection of < 5 

ppm. NTP (2005) reanalyzed sample NTP AQ-OX and reported a purity of ~ 99.8 and 0.09% 9-

NA contaminant (Sample A07496 from Study 2). 

METHOD

Industry accepted standard protocols and published procedures were followed in compliance 

with Good Laboratory Practice regulations (Ames et al., 1975; Brusick et al., 1980; Maron and 

Ames, 1983). 

Test type: Bacterial Reverse Mutation Test
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GLP compliant?: Yes 

Year study performed: 2001 (date of published journal article)

Test species: Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 and 

Escherichia coli strain WP2 uvrA.

Metabolic activation: With and without activation

Concentrations tested: 0, 30, 60, 125, 250, 500, 1000, and 2000 μg/plate 

Statistical methods used: Not specified

Test method/conditions remarks: “Analytical techniques including HPLC and GC-MS were 

performed to verify that the main component in all preparations was AQ, that purification did not 

alter the parent compound, and to identify potential contaminants. GC-MS analysis was 

conducted to identify impurities. U.S. EPA Methods 610, 625, and 8270 in the Code of Federal 

Register 40 identify 16 PAH and 72 additional toxic or carcinogenic substances of concern in 

environmental samples. Analyses were done using the conditions and looking for the target 

toxic compounds specified in these standard procedures. Quantitation of contaminants was 

based on HPLC analysis.” 

Salmonella-Escherichia coli/Mammalian-Microsome Reverse Mutation Assay with a 

confirmatory assay. The assay tested for the ability to induce reverse mutations at the histidine 

locus in the genomes of specific Salmonella typhimurium tester strains and at the tryptophan 

locus in an Escherichia coli tester strain in both the absence and presence an exogenous 

metabolic activation system provided by microsomal enzyme preparations derived from S-9 

(9000 g supernatant) fractions from the livers of Aroclor 1254-induced rats. The concentration 

levels used were based on a toxicity range-finding study. At the highest concentrations, 

precipitate of the test chemical was seen on the plates. No appreciable toxicity was observed 

with any of the samples tested. 

Results were presented as the mean number of revertant colonies per plate and the standard 

deviation of the mean. Concurrent positive and solvent controls were run with every trial.

Strain text description: TA98, TA100, TA1535, TA1537, and WP2 uvrA

Vehicle used: Yes

Vehicle Name: Unknown

Number of Replicates Used: Unknown

Criteria for Positive Results: “Criteria for a positive response were at least a 2-fold increase in 

the mean revertants per plate of at least one tester strain over the mean revertants per plate of the 
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appropriate vehicle control for test strains TA98, TA100 and WP2 uvrA and/or at least a 3-fold 

increase for tester strains TA1535 and TA1537.” 

Positive Control Substance Remarks: Without metabolic activation: 2-nitrofluorene at 1.0 

μg/plate in TA98, sodium azide at 2.0 μg/plate in TA100 and TA1535, 2-methoxy-6-chloro-9-

(2-chloroethylaminopropylamino)acridine dihydrochloride (ICR-191) at 2.0 μg/plate in TA1537, 

and 4-nitroquinoline-N-oxide at 1.0 μg/plate in WP2 uvrA. With metabolic activation: 

benzo[a]pyrene (B[a]P) at 2.5 μg/plate in TA98, 2-aminoanthracene at 2.5 μg/plate in TA100, 

TA1535, and TA1537, and 2-aminoanthracene at 25 μg/plate in WP2 uvrA.

The vehicle control substance was not specified.

RESULTS 

Genotoxicity result: Negative with and without metabolic activation for samples without 

detectible 9-NA contaminant; positive without metabolic activation and negative with metabolic 

activation for sample with 0.12% 9-NA contaminant (NTP AQ-OX; Sample A07496 from Study 

2).

Cytotoxic concentration (if applicable): Not specified

Results remarks: Positive and negative controls responded appropriately. “The NTP AQ-OX 

sample was mutagenic in a dose-dependent manner in strains TA98, TA100 and TA1537. 

Mutagenic activity was reduced or eliminated by addition of an S9 rat liver microsome metabolic 

activation system.” In contrast, neither purified NTP AQ-OX, AQ-FC nor AQ-DA showed any 

mutagenic activity either with or without metabolic activation in the Ames tester strains or in 

WP2uvrA. “Because AQ is not mutagenic, all the mutagenic activity in the NTP AQ-OX sample 

can be ascribed to the contaminant 9-NA. Mutagenic activity of the 9-NA is seen at 

concentrations as low as 0.15 μg/plate, indicating potent activity. In tester strain TA98, the 

induced revertants over controls per microgram of chemical for the concurrently run positive 

controls are 137 revertants/μg for benzo[a]P and equal in potency to 2-nitrofluorene.” 

CONCLUSIONS

“The chemical structure of AQ does not suggest that the parent compound would be DNA 

reactive. Therefore, a mutagenic contaminant was present in the NTP bioassay sample that is 

either directly mutagenic or can be activated by bacterial metabolism. Analytical studies showed 

that the primary contaminant 9-nitroanthracene (9-NA) was present in the NTP AQ-OX at a 

concentration of 1200 p.p.m., but not in the purified material. The 9-NA and any other 

contaminants that might have been present in the NTP AQ-OX induced measurable mutagenicity 

at 9-NA concentrations as low as 0.15 μg/plate in tester strain TA98, indicating potent mutagenic 

activity. On the basis of revertants per microgram, 9-NA was more potent than B[a]P and was 

about equally as potent as the 2-nitrofluorene run concurrently as positive controls.”

According to Butterworth et al. (2001), “the report of direct-acting genotoxic activity in two 

Ames tests (Liberman et al., 1982; Zeiger et al., 1988) was unexpected because the data were in 
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conflict with so many other reports of negative mutagenic activity.” However, both studies were 

conducted on samples of AQ that had a purity of only 97% and analysis of the identity of the 

remaining 3% of non-AQ material was not done.

STUDY RELIABILITY

[2] Reliable with restrictions. Important study details were missing including solvent control 

substance, test conditions (duration and temperature of incubation), rationale for selection of test

concentrations, media used, treatment procedures, number of replicates, statistical analyses, 

individual plate counts, and detailed results. 

REFERENCES

Butterworth, B.E., Mathre, O., and Ballinger, K. 2001. The preparation of anthraquinone used 

in the National Toxicology Program cancer bioassay was contaminated with the mutagen 9-

nitroanthracene. Mutagenesis 16(2):169-177. Text in quotes is taken from Butterworth et al. 

(2001).
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Study 2: Mutagenicity study in Salmonella typhimurium (NTP, 2005)

Title: Toxicology and carcinogenesis studies of anthraquinone (CAS No. 84-65-1) in F344/N 

rats and B6C3F1 mice (feed studies).

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: 9,10-Anthracenedione, CASRN 84-65-1 (commonly called anthraquinone)

Remarks: “Anthraquinone (97% pure) was manufactured by Aldrich Chemical Company and 

sent to the laboratory as a coded aliquot from Radian Corporation (Austin, TX). Anthraquinone 

(100% pure) was sent to the study laboratory as coded aliquots from Battelle Columbus 

Operations (Columbus, OH). Since February 2004, tests were conducted with four 

anthraquinone samples provided by industry and selected to represent a broad sampling of 

anthraquinones produced by different manufacturing processes1. These samples of 

9,10-anthraquinone were provided by Zeneca Fine Chemicals (Wilmington, DE), Environmental 

Biocontrol International (Wilmington, DE), or Kawasaki Kasei Chemicals, Ltd. (Kawasaki City, 

Kanagawa, Japan), via Environmental Biocontrol International and sent to the laboratory as 

coded aliquots.”

Overall, NTP (2005) and Zeiger et al. (1988) tested the following six samples:

(1) AQ (97% pure); (2) AQ (100% pure); (3) AQ Sample A07496 (Lot No. 5893) from Zeneca 

Fine Chemicals produced using the nitric acid oxidation process and reported to be 99.8% pure 

based on reanalysis by NTP (Butterworth et al., 2001 had reported a purity of 99% for this 

sample (NTP AQ-OX)2

). This sample was from the lot used in the NTP 2-year studies. In an 

analysis by NTP, the primary contaminant in this sample was determined to be 0.09% 9-NA; (4) 

AQ Sample A65343 (Lot No. 64005) from Environmental Biocontrol International was produced 

using the Diels-Alder process; (5) AQ Sample A54984 (Lot No. GSTU 2517770) from 

Environmental Biocontrol International was produced using the Friedel-Crafts process; (6) AQ 

Sample A40147 (Lot No. 2Y011) was produced by Kawasaki Kasei Chemical, Ltd., using the 

Diels-Alder process and was approximately 99.4% pure. The primary contaminant of Sample 

A40147 was 9-fluorenone (CAS No. 486-25-9), which has been reported to be inactive in two 

bacterial mutagenicity assays (Vasilieva et al., 1990). 9-Fluorenone has not been tested for 

genotoxicity by the NTP.

METHOD

Method/guideline followed: NTP Protocols

 

1

 “There are three methods used for the commercial synthesis of anthraquinone: a Friedel-Crafts reaction between 

benzene and phthalic anhydride, a Diels-Alder synthesis from 1,4-naphthoquinone and 1,3-butadiene, and the 

oxidation of anthracene in acid.”

2 See summary for Study 1 in Section C-6.
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Test type: Bacterial Reverse Mutation Test

GLP compliant?: Not specified

Year study performed: 

(1) AQ (97% pure): 1985

(2) AQ (100% pure): 2003

(3) AQ (A07496, 99.8% pure): 2004

(4) AQ (A65343, Diels-Alder): 2005

(5) AQ (A54984, Friedel-Crafts): 2004 

(6) AQ (A40147, Diels-Alder, 99.4%): 2006

Test species: “All samples were tested in Salmonella strains TA98 and TA100; 100% pure 

anthraquinone was also tested in TA102, and Sample A07496 was also tested in TA1537. Strain 

1537 was added in order to independently test the 2-year bioassay sample in the same strains that 

gave positive results in the Butterworth et al. (2001) study.”

Metabolic activation: With and without S9

Concentrations tested: 

(1) AQ (97% pure): 0, 33, 100, 333, 1000, and 2500 μg/plate

(2) AQ (100% pure): 0, 100, 333, 1000, 3333, and 10,000 μg/plate

(3) AQ (A07496, 99.8% pure): 0, 30, 100, 300, 1000, 3000, and 10,000 μg/plate

(4) AQ (A65343, Diels-Alder): 0, 30, 100, 300, 1000, 3000, and 10,000 μg/plate

(5) AQ (A54984, Friedel-Crafts): 0, 30, 100, 300, 1000, 3000, and 10,000 μg/plate

(6) AQ (A40147, Diels-Alder, 99.4%): 0, 30, 100, 300, 1000, 3000, and 10,000 μg/plate

Statistical methods used: None stated

Test method/conditions remarks: “The samples were incubated with the Salmonella 

typhimurium tester strains either in buffer or S9 mix (metabolic activation enzymes and cofactors 

from Aroclor 1254-induced male Sprague-Dawley rat or Syrian hamster liver) for 20 minutes at 

37C. After the 20-minute preincubation period, top agar supplemented with L-histidine and 

d-biotin was added, and the contents of the tubes were mixed and poured onto the surfaces of 

minimal glucose agar plates. Histidine-independent mutant colonies arising on these plates were 

counted following incubation for 2 days at 37C. Each trial consisted of triplicate plates of 

concurrent positive and negative controls and at least five doses of each sample.” 

“In general, samples were tested with 10% rat S9; Sample A07496 was also tested by the NTP 

with 30% rat S9 because it had tested the 97% pure anthraquinone sample with 30% rat S9.”

Positive Control Substances: Without metabolic activation: Sodium azide (TA100), 

9-aminoacridine (TA1537), 4-nitro-o-phenylenediamine (TA98), and mytomycin-C (TA102)

With metabolic activation: 2- aminoanthracene (all strains)
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Solvent Control Substance: Dimethylsulfoxide for 97% pure anthraquinone and Samples 

A65343, A54984, and A40147; Propylene glycol for 100% pure anthraquinone; Dimethyl 

sulfoxide and propylene glycol for Sample A07496

Criteria for Positive Response: “In this assay, a positive response is defined as a reproducible, 

dose-related increase in histidine-independent (revertant) colonies in any one strain/activation 

combination. An equivocal response is defined as an increase in revertants that is not dose 

related, is not reproducible, or is not of sufficient magnitude to support a determination of 

mutagenicity. A negative response is obtained when no increase in revertant colonies is 

observed following chemical treatment. There is no minimum percentage or fold increase 

required for a chemical to be judged positive or weakly positive.”

RESULTS 

Genotoxicity result: Equivocal

(1) AQ (97% pure) Positive in TA98 and TA100 with and without S9

(2) AQ (100% pure) Negative in TA98, TA100, TA102 with and without S9

(3) AQ (A07496, 99.8% pure) Negative in TA98, TA100, TA1537 with and without S9

(4) AQ (A65343, Diels-Alder) Negative in TA98, TA100 with and without S9

(5) AQ (A54984, Friedel-Crafts) Negative in TA98, TA100 with and without S9

(6) AQ (A40147, Diels-Alder 99.4%) Positive in TA98, TA100 with and without S9

Cytotoxic concentration (if applicable): No data

Results remarks: “Anthraquinone (97% pure) (33 to 2,500 μg/plate) was mutagenic in 

Salmonella typhimurium strains TA98 and TA100, with and without 30% hamster and rat liver 

S9 enzymes (Zeiger et al., 1988). A 100% pure sample of anthraquinone (100 to 10,000 

μg/plate) showed no detectable mutagenic response in TA98, TA100, or TA102, with or without 

10% rat S9. Sample A07496, the compound used in the 2-year NTP studies (99.8% pure), was 

negative in TA98, TA100, and TA1537, with and without 10% and 30% rat S9 at concentrations 

up to 10,000 μg/plate. Samples A65343 (Diels-Alder process) and A54984 (Friedel-Crafts 

process) were negative in TA98 and TA100, with and without 10% rat S9 at concentrations up to 

10,000 μg/plate. Sample A40147 (Diels-Alder process) was mutagenic in TA98 and TA100, 

with and without 10% rat S9. The lowest effective doses in TA98 for Sample A40147 were 

100 μg/plate without S9 and 1,000 μg/plate with S9. The response in TA100 was less 

impressive; the lowest effective doses were 10,000 μg/plate without S9 and 3,000 μg/plate with 

S9. The highest dose tested, 10,000 μg/plate, is higher than those most laboratories use in the 

absence of dose limiting toxicity.” Positive and solvent controls responded appropriately.

CONCLUSIONS

Two out of six samples of anthraquinone induced gene mutations in bacteria with and without 

metabolic activation.
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STUDY RELIABILITY

[1] Reliable without restrictions. 

REFERENCES

NTP (National Toxicology Program). 2005. Toxicology and carcinogenesis studies of 

anthraquinone (CAS No. 84-65-1) in F344/N rats and B6C3F1 mice (feed studies). NTP TR494. September, 2005. Text in quotes is taken from NTP (2005).

Butterworth, B., Mathre, O., and Ballinger, K. 2001. The preparation of anthraquinone used in 

the National Toxicology Program cancer bioassay was contaminated with mutagen 

9-nitroanthracene. Mutagenesis 16(2):169-177.

Zeiger, E., Anderson, B., Haworth, S., Lawlor, T., and Mortelmans, K. 1988. Salmonella 

mutagenicity tests: IV. Results from the testing of 300 chemicals. Environ. Mol. Mutagen. 

11(Suppl. 12): 1-158.
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Study 3: L5178Y thymidine kinase (TK) +/- mouse lymphoma forward mutation assay 

(Butterworth et al., 2001)

Title: The preparation of anthraquinone used in the National Toxicology Program cancer 

bioassay was contaminated with the mutagen 9-nitroanthracene.

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: 9,10-Anthracenedione, CASRN 84-65-1 (commonly called anthraquinone)

Remarks: Supplied by Environmental Biocontrol, Intl (Wilmington, DE); According to GC-MS 

analyses, the sample AQ-DA was 99% pure and the contaminant 9-NA was not detected at a 

limit of detection of < 50 ppm 

METHOD

Method/guideline followed: Industry accepted standard protocols and published procedures 

were followed in compliance with Good Laboratory Practice regulations (Amacher et al., 1980; 

Clive et al., 1987).

Test type: Mouse Lymphoma Forward Mutation Assay

GLP compliant?: Yes

Year study performed: 2001 (date of published journal article)

Test species: Mouse lymphoma L5178Y cell line

Metabolic activation: With and without

Concentrations tested: 

Without metabolic activation: 0 (control), 3.13, 6.25, 12.5, 25, 37.5 and 50 μg/mL

With metabolic activation: 0 (control), 1.57, 3.13, 6.25, 12.5, 25, 37.5 and 50 μg/mL

Statistical methods used: None specified

Test method/conditions remarks: “A sample of AQ-DA was submitted to Covance 

Laboratories (Vienna, VA) to test for mutagenic activity in the L5178Y TK +/- mouse 

lymphoma forward mutation assay. The objective of the assay was to evaluate the ability of AQ 

to induce forward mutations at the TK locus in the mouse lymphoma L5178Y cell line. The test 

article formed a suspension in dimethylsulfoxide at concentrations > 1.56 mg/kg [sic] and was 

insoluble in medium above ~ 25 μg/mL. Assays were run with and without a rat liver S9 

metabolic activation system. Range-findings studies showed AQ to be non-toxic at nominal 

doses up to 500 μg/mL with and without metabolic activation. The testing limit for the mutation 
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assays was set at 50 μg/mL, which is about twice the solubility limit in medium. Two 

independent assays were conducted each with and without metabolic activation. The criterion 

for a positive response is induction of a mutation frequency that is at least two times that of the 

control mutant frequency for that given experiment. Colony sizing was not done because no 

positive responses were observed with AQ.” 

Positive Control Substance: 5 nL/mL methylmethanesulfonate without metabolic activation; 

2 μg/mL methylcholanthrene with metabolic activation

RESULTS

Genotoxicity result: Negative with and without metabolic activation

Cytotoxic concentration (if applicable): Nontoxic up to 500 μg/mL with and without 

metabolic activation 

Results remarks: “No cytotoxicity was observed in any of the trials in the L5178Y mutation 

assays. Mutant frequencies of treated cultures varied randomly with dose toxicity and no 

increases above the minimum criteria for a positive response were induced. AQ-DA was 

therefore evaluated as negative with and without metabolic activation at the TK locus in L5178Y 

mouse lymphoma cells under the conditions used in this study.”

CONCLUSIONS

The test chemical did not induce forward gene mutations in L5178Y mouse lymphoma cells 

either with or without metabolic activation.

STUDY RELIABILITY

[2] Reliable with restrictions. Important study details were missing including test guidelines 

followed, statistical methods used, negative control treatment, number of replicates, and volume 

of test substance added. 

REFERENCES

Butterworth, B., Mathre, O., and Ballinger, K. 2001. The preparation of anthraquinone used in 

the National Toxicology Program cancer bioassay was contaminated with mutagen 

9-nitroanthracene. Mutagenesis 16(2):169-177. Text in quotes is taken from Butterworth et al. 

(2001).
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C-7. Genetic Toxicity – Chromosomal Aberrations

Summary of endpoint: Adequate 

Study 1: In vitro chromosomal aberration induction in Chinese hamster ovary (CHO) cells 

(Butterworth et al., 2001)

Title: The preparation of anthraquinone used in the National Toxicology Program cancer 

bioassay was contaminated with the mutagen 9-nitroanthracene.

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: 9,10-Anthracenedione, CASRN 84-65-1 (commonly called anthraquinone)

Remarks: Supplied by Environmental Biocontrol, Intl (Wilmington, DE); According to GC-MS 

analyses, the sample AQ-DA was 99% pure and the contaminant 9-NA was not detected at a 

limit of detection of < 50 ppm 

METHOD

Method/guideline followed: Industry accepted standard protocols and published procedures 

were followed in compliance with Good Laboratory Practice regulations (Evans, 1962)

Test type: Chromosomal Aberrations in Mammalian Cells (in vitro)

GLP compliant?: Yes

Year study performed: 2001 (date of published journal article)

Test species: Chinese hamster ovary (CHO) cells

Metabolic activation: With and without

Concentrations tested: 0 (control), 12.5, 25, 37.5, and 50 μg/mL

Statistical methods used: None specified 

Test method/conditions remarks: “A sample of AQ-DA was submitted to Covance 

Laboratories (Vienna, VA) to test for the ability to induce chromosomal aberrations in CHO cells 

with and without metabolic activation. A dose range-finding study was conducted to select 

experimental doses. Solubility considerations determined the highest dose to be 50 μg/mL. 

Replicate cultures of CHO cells were incubated with up to 50 μg/mL AQ-DA with and without 

metabolic activation, with a 20.0 h harvest in an initial trial and with 20.0 and 44.0 harvests in 

the confirmatory trials. No visual signs of cytotoxicity were observed in the cultures analyzed. 
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A test article was considered positive for induction chromosomal aberrations if a significant 

increase was observed compared to controls at a level of P < 0.01.”

Positive Control Substance: 0.1 μg/mL Mytomycin [sic] C for 20-hour exposure without 

metabolic activation; 5.0 μg/mL cyclophosphamide (CP) for 20 h exposure with metabolic 

activation.

RESULTS

Genotoxicity result: Negative with and without metabolic activation

Cytotoxic concentration: No data

Results remarks: “No significant increase in cells with chromosomal aberrations, polyploidy or 

endoreduplication was observed at the concentrations analyzed. AQ-DA was considered 

negative for inducing chromosomal aberrations in CHO cells with and without metabolic 

activation.” 

CONCLUSIONS

The test chemical does not appear to have the potential to induce chromosomal aberrations in 

CHO cells in vitro.

STUDY RELIABILITY

[2] Reliable with restrictions. Important study details were missing including test guidelines 

followed, statistical methods used, test conditions, negative control treatment, number of 

replicates, cytotoxicity in main experiment, individual culture data, source and details of cell line 

tested, and detailed numerical results.

REFERENCES

Butterworth, B., Mathre, O., and Ballinger, K. 2001. The preparation of anthraquinone used in 

the National Toxicology Program cancer bioassay was contaminated with mutagen 

9-nitroanthracene. Mutagenesis 16(2):169-177. Text in quotes is taken from Butterworth et al. 

(2001).
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Study 2: In vivo bone marrow mouse micronucleus assay (Butterworth et al., 2001)

Title: The preparation of anthraquinone used in the National Toxicology Program cancer 

bioassay was contaminated with the mutagen 9-nitroanthracene

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: 9,10-Anthracenedione, CASRN 84-65-1 (commonly called anthraquinone)

Supplied by Environmental Biocontrol, Intl. (Wilmington, DE); According to GC-MS analyses, 

the sample AQ-DA was 99% pure and the contaminant 9-NA was not detected at a limit of 

detection of < 50 ppm.

METHOD

Method/guideline followed: Industry accepted standard protocols and published procedures 

were followed in compliance with Good Laboratory Practice regulations (Heddle et al., 1991; 

Salamone and Mavourin, 1994; Schmid, 1976).

Test type: Mammalian Erythrocyte Micronucleus Test (in vivo)

GLP compliant?: Yes 

Year study performed: 2001 (date of published journal article)

Test species: Crl:CD-1 (ICR) BR mice (males and females) 

Number of animals/sex/dose: 5/sex/dose/harvest time 

Route of administration: Oral − gavage 

Vehicle: Corn oil 

Exposure duration: Following a single dose, animals were killed at 24, 48, and 72 hours for 

extraction of bone marrow.

Doses tested: 1250, 2500, and 5000 mg/kg

Statistical methods used: None specified

Test method/conditions remarks: “The objective of this whole animal assay was to evaluate 

the ability of AQ-DA to induce micronuclei in bone marrow polychromatic erythrocytes (PCE) 

of Crl:CD-1 (ICR) BR mice. In the dose selection study the test article was suspended in corn 

oil and dosed by oral gavage at up to 5000 mg/kg. Based on lack of significant toxicity, 5000 
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mg/kg was selected as the highest dose for the micronucleus studies. In the micronucleus assay, 

AQ-DA was suspended in corn oil and dosed by oral gavage at 1250, 2500 and 5000 mg/kg. 

Five males and five females were randomly assigned to each dose per harvest time group. 

Animals were killed ~ 24, 48 and 72 h after dosing with the test article for extraction of the bone 

marrow.” 

Bone marrow was extracted and slides were prepared according to accepted procedures so that 

they could be examined for the presence of micronuclei in bone marrow polychromatic 

erythrocytes (PCEs). The ratio of polychromatic erythrocytes to normochromatic erythrocytes 

(PCE:NCE) was calculated to see if there was bone marrow toxicity. One thousand PCEs were 

examined per mouse and the results for each group were presented as the mean for five animals 

and its standard error. 

Replicates: 3

Positive Control Substance: Cyclophosphamide at 80 mg/kg

Negative Control Substance Remarks: The vehicle control was corn oil.

Criteria for Positive Results: A response was judged positive if it was significantly greater than 

the corresponding vehicle control at a level of P < 0.01. 

RESULTS

Genotoxicity result: Negative

Cytotoxic dose: No data

Results remarks: The PCE:NCE ratio did not decrease to give any indication of bone marrow 

toxicity. The test chemical did not induce a significant increase in micronuclei in bone marrow 

PCEs under the conditions of this assay. The positive and vehicle controls gave appropriate 

responses. None of the experimental groups approached being statistically significantly higher 

than the corresponding vehicle control. The authors discussed other reports of results using this 

or similar assays. They cited an NTP study in 1999 that gave a negative result using the test 

chemical as prepared by oxidation of anthracene (AQ-OX). When the chemical is prepared that 

way, it is sometimes contaminated with strong mutagens, as summarized in the Robust Study 

Summary for this chemical for the gene mutation assay. They also discussed an NTP (1999) 

mouse peripheral blood micronuclei assay from a 14-week range-finding study that yielded the 

tentative conclusion that AQ-OX exhibited weak activity for inducing micronuclei. Butterworth 

et al. (2001) stated that the doses used [in that earlier assay] were up to four times the maximum 

tolerated dose used in the [following cancer] bioassay, no response was seen in the female 

animals, and the response in the males was judged as positive only with a highly nonconservative trend test.
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CONCLUSIONS

The test chemical does not appear to have the potential to induce micronuclei formation in bone 

marrow polychromatic erythrocytes in vivo.

STUDY RELIABILITY

[2] Reliable with restrictions. Important study details were missing including test guidelines 

followed, individual animal data, statistical methods, details of test substance preparation, bone 

marrow preparation and analysis, data from range-finding study, and detailed results. 

REFERENCES

Butterworth, B., Mathre, O., and Ballinger, K. 2001. The preparation of anthraquinone used in 

the National Toxicology Program cancer bioassay was contaminated with mutagen 

9-nitroanthracene. Mutagenesis 16(2):169-177. Text in quotes is taken from Butterworth et al. 

(2001).
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Study 3: In vivo mouse bone marrow micronucleus test (NTP, 2005)

Title: Toxicology and carcinogenesis studies of anthraquinone (CAS No. 84-65-1) in F344/N 

rats and B6C3F1 mice (feed studies).

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: 9,10-Anthracenedione, CASRN 84-65-1 (commonly called anthraquinone)

Remarks: Purity not specified 

METHOD

Method/guideline followed: Shelby et al. (1993), cited in NTP (2005)

Test type: Mammalian Erythrocyte Micronucleus Test (in vivo)

GLP compliant?: Not specified

Year study performed: 1989

Test species: B6C3F1 male mice

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

Route of administration: Intraperitoneal 

Vehicle: Corn oil

Exposure duration: 72 hours

Doses tested: 0, 500, 1000, and 2000 mg/kg 

Statistical methods used: “The results were tabulated as the mean of the pooled results from all 

animals within a treatment group plus or minus the standard error of the mean. The frequency of 

micronucleated cells among PCEs was analyzed by a statistical software package that tested for 

increasing trend over dose groups with a one-tailed Cochran-Armitage trend test, followed by 

pairwise comparisons between each dosed group and the control group (ILS, 1990). In the 

presence of excess binomial variation, as detected by a binomial dispersion test, the binomial 

variance of the Cochran-Armitage test was adjusted upward in proportion to the excess 

variation.”

Test method/conditions remarks: “Male B6C3F1 mice were injected intraperitoneally three 

times at 24-hour intervals with anthraquinone dissolved in corn oil. The animals were killed 
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24 hours after the third injection, and smears were prepared from bone marrow cells obtained 

from the femurs. Air-dried smears were fixed and stained; 2,000 polychromatic erythrocytes 

(PCEs) were scored for the frequency of micronucleated cells in each of five animals per dose 

group.”

Positive Control Substance: Dimethylbenzanthracene at 12.5 mg/kg

Negative Control Substance: Corn oil at 0.4 mL per injection 

One limitation of this experiment was that there was no testing of females. The testing in 

females becomes much less important when toxicity is similar between the sexes, and there were 

ample data available within the same report to show that such is the case. However, this issue 

was never dealt with in the presentation regarding this assay.

Criteria for Positive Results: “In the micronucleus test, an individual trial is considered positive 

if the trend test P value is less than or equal to 0.025 or if the P value for any single dose group is 

less than or equal to 0.025 divided by the number of dose groups. A final call of positive for 

micronucleus induction is preferably based on reproducibly positive trials (as noted above). 

Ultimately, the final call is determined by the scientific staff after considering the results of 

statistical analyses, the reproducibility of any effects observed, and the magnitudes of those 

effects.”

RESULTS

Genotoxicity result: Negative

Cytotoxic dose: No data

Results remarks: The test chemical did not induce a statistically significant increase in 

micronuclei in bone marrow PCEs under the conditions of this assay. The positive and vehicle 

controls gave appropriate responses. 

CONCLUSIONS

The test chemical does not appear to have the potential to induce the formation of micronuclei in 

bone marrow PCEs in vivo.

STUDY RELIABILITY

[2] Reliable with restrictions. The purity and composition of the test substance were not 

specified.

REFERENCES

NTP (National Toxicology Program). 2005. Toxicology and carcinogenesis studies of 

anthraquinone (CAS No. 84-65-1) in F344/N rats and B6C3F1 mice (feed studies). NTP TR494. September, 2005. Text in quotes is taken from NTP (2005).
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Shelby, M.D., Erexson, G.L., Hook, G.J., and Tice, R.R. 1993. Evaluation of a three-exposure 

mouse bone marrow micronucleus test protocol: Results with 49 chemicals. Environ. Mol. 

Mutagen. 21:160-179.
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Study 4: In vivo mouse peripheral blood micronucleus test (NTP, 2005)

Title: Toxicology and carcinogenesis studies of anthraquinone (CAS No. 84-65-1) in F344/N 

rats and B6C3F1 mice (feed studies).

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: 9,10-Anthracenedione, CASRN 84-65-1 (commonly called anthraquinone)

Remarks: ~ 99.8% pure; Lot No. 5893; Supplied by Zeneca Fine Chemicals (Wilmington, DE)

METHOD

Method/guideline followed: MacGregor et al. (1990), cited in NTP (2005)

Test type: Mammalian Erythrocyte Micronucleus Test (in vivo)

GLP compliant?: Yes

Year study performed: 1994

Test species: B6C3F1 mice (male and female)

Number of animals/sex/dose: 5

Route of administration: Oral – dietary

Vehicle: No

Exposure duration: 14 weeks

Doses tested: 0, 1875, 3750, 7500, 15,000, and 30,000 ppm (equivalent to average daily doses 

of approximately 250, 500, 1050, 2150, and 4300 mg/kg for males and 300, 640, 1260, 2600, and 

5300 mg/kg for females, respectively)

Statistical methods used: None specified

Test method/conditions remarks: This study is a continuation of the NTP 14-week dietary 

study in mice described above. “At the end of the 14-week study, peripheral blood samples were 

obtained from male and female B6C3F1 mice. Smears were immediately prepared and fixed in 

absolute methanol. The methanol-fixed slides were stained with acridine orange and coded. 

Slides were scanned to determine the frequency of micronuclei in 2,000 normochromatic 

erythrocytes (NCEs) and the percent of polychromatic erythrocytes (PCEs) in 1,000 total 
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erythrocytes in each of five animals per exposure group. The percent PCEs among the entire 

erythrocyte population was determined as a measure of bone marrow toxicity.

The results were tabulated and the frequency of micronucleated cells among NCEs was analyzed 

as described for PCEs in the bone marrow micronucleus test protocol.”

Criteria for Positive Results: “Pairwise comparison with the vehicle control; significant at P ≤ 

0.005 (ILS, 1990). Significance of micronucleated NCEs/1,000 NCEs tested by the one-tailed 

trend test; significant at P ≤ 0.25 (ILS, 1990).”

RESULTS

Genotoxicity result: Positive

Cytotoxic dose: No data

Results remarks: “Significant increases over the control frequencies were noted in male and 

female mice at the highest exposure concentration. Although only the 30,000 ppm female group 

differed significantly from the control frequency by pairwise comparison, both data sets yielded 

positive trend tests, and the peripheral blood micronucleus test was judged to be positive for both 

male and female mice. Evidence of increased erythropoiesis in treated mice was demonstrated 

by the slightly elevated percent polychromatic erythrocyte (PCE) values in several of the 

exposure groups, mostly in exposed female mice. The data do not demonstrate a direct 

correlation between percent PCE values and micronucleus frequency except in the high exposure 

concentration groups where both male and female mice showed the highest frequencies of 

micronucleated erythrocytes and the highest percent PCE values. An increased rate of 

erythropoiesis may have contributed to the micronucleus responses seen in the high exposure 

concentration groups, because increased cell proliferation can produce increased levels of mitotic 

errors.”

CONCLUSIONS

The test chemical appears to have the potential to induce micronuclei formation in peripheral 

blood erythrocytes in vivo.

STUDY RELIABILITY

[1] Reliable without restrictions. 

REFERENCES

NTP (National Toxicology Program). 2005. Toxicology and carcinogenesis studies of 

anthraquinone (CAS No. 84-65-1) in F344/N rats and B6C3F1 mice (feed studies). NTP TR494. September, 2005. Text in quotes is taken from NTP (2005).
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MacGregor, J.T., Wehr, C.M., Henika, P.R., and Shelby, M.D. 1990. The in vivo erythrocyte 

micronucleus test: Measurement at steady state increases assay efficiency and permits 

integration with toxicity studies. Fundam. Appl. Toxicol. 14:513-522.
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C-8. Reproductive/Developmental Toxicity

Summary of endpoint: Adequate

Study 1: A reproductive/developmental toxicity study in rats (MPI Research, Inc., 2012)

Title: An oral reproduction/developmental toxicity screening study of anthraquinone (CAS #84-

65-1) in rats

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: 9,10-Anthracenedione (CASRN 84-65-1)

Remarks: 99.4% purity; Lot No. 1543-I-GESU5099797, sampled from a commercial shipment; 

manufactured by the Friedel-Crafts process

METHOD

Method/guideline followed: OECD TG 421

Test type: Reproduction/developmental toxicity screening test

GLP compliant?: Yes

Year study performed: 2006

Test species: Sprague-Dawley CD[Crl:CD(SD)] rat; age: ~ 8 weeks old; initial body weight: 

237-263 g (males) or 186-208 g (females)

Number of animals/sex/dose: 12/sex/dose

Route of administration: Oral gavage

Exposure duration: 14 days prior to mating, during mating (up to 14 days), and for 

14 additional days for males or through lactation day 4 for females 

Doses/concentrations tested: Nominal: 0, 150, 600, and 2400 mg/kg-bw/day; Mean measured: 

88, 405, and 2028 mg/kg-bw/day

Vehicle: 1% guar gum in distilled water

Statistical methods used: Treatment effects were analyzed using either analyses of variance 

(ANOVA) or Fisher’s exact test. For parameters analyzed by ANOVA, if sample sizes for all 

groups were 3 or greater, Levene's test was used to assess homogeneity of group variances for 
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each specified endpoint and for all collection intervals. If Levene's test was not significant (P > 

0.01), a pooled estimate of the variance (Mean Square Error or MSE) was computed from a oneway ANOVA and pairwise comparisons were made using a Dunnett's test to compare each 

treatment group with the control group. If there were only two groups involved, the Dunnett's 

test reduced to a Student's t-test. If Levene's test was significant (P < 0.01), comparisons with 

the control group were made using Welch's t-test with a Bonferroni correction. Groups with 

sample sizes less than 3 were excluded from statistical analysis. Data comprised of percent 

values were transformed using the arcsin of the square root before data analysis.

For parameters analyzed by Fisher’s exact test, an overall test for association between response 

and treatment was conducted. If this overall test was significant (P < 0.05) and there were more 

than two groups, then a follow-up analysis was conducted to compare each treatment group to 

the control group. 

To determine effects of treatment on pup body weight, an analysis of covariance was conducted 

on mean pup body weight per dam using litter size as a covariate. Pairwise comparisons were 

conducted using Dunnett’s test.

Test method/conditions remarks: To prepare the test article formulations at each

concentration, the required amount of prepared vehicle (guar gum) was measured and transferred 

into a beaker. The required amount of test article (CASRN 84-65-1) was weighed and slowly 

transferred into the beaker containing the vehicle, while mixing with a Silverson homogenizer. 

The contents of the beaker were mixed until uniform in appearance. The contents of the beaker 

were transferred into a graduated cylinder. Vehicle was added to the cylinder to yield the 

required volume of prepared test article formulation. The cylinder was inverted to thoroughly 

mix the contents, which were then transferred into a beaker. The contents of the beaker were 

stirred using a motor-driven propeller and evenly dispensed into amber glass containers, while 

stirring, using a syringe. Formulations of the test article were prepared weekly for each 

concentration, and were stored refrigerated. On occasion, additional preparations were made as 

necessary during the course of the study. Two test batches were mixed prior to study initiation.

To assess the homogeneity of the test article preparations, samples (2.0 mL) of the dosing 

formulations were collected at the 150 and 2400 mg/kg-bw/day nominal dose levels from each 

test batch and from the third formulation preparation. Duplicate samples were collected from the 

top, middle, and bottom of the container, while stirring, using a syringe. 

Samples (2.0 mL) of the dosing formulations at each concentration were collected from Weeks 1, 

3, and 8 to assess the concentration of the test article at each dose level. The samples were 

collected from the middle of the container, while stirring, using a syringe. Samples were 

analyzed using an HPLC system with UV detector.

Sprague-Dawley rats (12/sex/dose) were administered the test substance via gavage at nominal 

dose levels of 0, 150, 600, or 2400 mg/kg-bw/day at a dose volume of 20 mL/kg. Control 

animals received the vehicle, 1% guar gum, in distilled water. Animals were exposed for 2 

weeks prior to mating and during mating (up to 14 days); males were exposed for an additional 2 
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weeks after mating and females were exposed through gestation and lactation day 4. Food and 

water were provided ad libitum during the study.

Observations of the animals included clinical signs, body weights, and food consumption. All 

animals were observed for morbidity, mortality, injury, and the availability of food and water 

twice daily throughout the duration of the study. Daily during treatment (approximately 

30-90 minutes post-dose), each animal was removed from the cage and given a detailed clinical

examination. Necropsy examinations were performed on all adult animals euthanized in 

extremis or found dead, and all surviving animals at the end of the exposure period; special 

emphasis was placed on the organs of the reproductive system. Organs weighed included the 

epididymis, prostate gland, seminal vesicles, testes, ovaries, and uterus with cervix. These same 

organs, as well as gross lesions, were examined microscopically in the control and high-dose 

animals. Implantation sites in the uterus were counted. 

The litters were examined as soon as possible after delivery and parameters including litter size, 

the sex of the pups, number of dead and live born pups, and any gross abnormalities of the pups 

were recorded. Any intact pups found dead at birth were externally examined; stillborn status 

was determined by lung floatation in water. Any intact pups found dead during lactation were 

externally examined. Pups were weighed, sexed, and examined for external abnormalities on 

lactation days 0 and 4. On lactation day 4, the pups were euthanized via intraperitoneal injection 

of sodium pentobarbital solution and examined for external abnormalities.

RESULTS

Endpoint value(s): The LOAEL for systemic toxicity was a nominal dose of 150 mg/kg-bw/day 

(88 mg/kg-bw/day calculated from measured concentrations) based on lower body weights in 

females and reduced food consumption in both males and females; a NOAEL was not 

established for systemic toxicity. The LOAEL for reproductive toxicity was a nominal dose of 

150 mg/kg-bw/day (88 mg/kg-bw/day calculated from measured concentrations) based on a 

decrease in the number of estrous cycles per animal; a NOAEL for reproductive toxicity was not 

established. The LOAEL for developmental toxicity was a nominal dose of 600 mg/kg-bw/day 

(405 mg/kg-bw/day calculated from measured concentrations) based on an increase in the 

number of stillborn pups, as well as decreases in the total number of pups delivered, number of 

liveborn pups, number of implantation scars per litter, pup body weight on lactation days 0 and 

4, and pup survival to lactation day 4; the NOAEL for developmental toxicity was a nominal 

dose of 150 mg/kg-bw/day (88 mg/kg-bw/day calculated from measured concentrations). 

Results remarks: Preparation of the dosing suspensions was problematic. Due to the viscosity 

of the vehicle (1% guar gum), the formulations could not be stirred adequately and, therefore, it 

was difficult to obtain uniform suspensions. Based on periodic concentration analysis of the test 

article suspensions prepared for study, the study authors noted that the formulations for the most 

part neither were homogeneous nor were the intended concentration levels achieved. 

As a measurement of homogeneity of dosing formulations, the testing laboratory provided the 

relative standard deviation (RSD) of the measured concentrations of samples taken from the top, 

middle, and bottom of the container. For the low dose, the RSD ranged from 12.0 to 26.5% for 
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the three batches of dosing formulations that were analyzed. For the high dose, the RSD ranged 

from 7.2 to 13.7%.

In test solution samples that were used to estimate test article concentration at each dose level, 

recoveries were 66.0-73.0% (Week 1), 59.5-84.7% (Week 3), and 25.1-99.5% (Week 8). Based 

on measured concentrations of the dosing formulations, the actual dose levels were 109, 396, and 

1661 mg/kg-bw/day for Week 1, 116, 357, and 2034 mg/kg-bw/day for Week 3, and 38, 463, and 

2388 mg/kg-bw/day for Week 8, respectively, for the 150, 600, and 2400 mg/kg-bw/day nominal 

dose levels. The mean dose levels (based on the average of measured concentrations from 

Weeks 1, 3, and 8) were 88, 405, and 2028 mg/kg-bw/day. No test article was detected in the 

control samples.

Test article-related mortality was seen in females at the mid- and high-dose levels. In the midand high-dose groups, 8/12 and 7/12 females, respectively, died or were euthanized in extremis; 

most of these mortalities occurred during the gestation and lactation periods. One male in the 

low-dose group died, but in the absence of mortality among males at the higher dose levels and 

since macroscopic findings were unremarkable, this death was considered unrelated to treatment 

with anthraquinone. 

Decreased body weights were observed in both males and females. Mean body weights of 

treated males were significantly lower than those of the controls throughout the mating and 

postmating periods at the mid- and high dose. Mean body weights of treated females at all dose 

levels were significantly lower than those of the controls during the premating, mating, gestation, 

and lactation periods. Gestational body weight gain was significantly lower in the mid- and 

high-dose groups for all intervals (gestation days 0-7, 7-14, 14-20, and 0-20), with the exception 

of the mid-dose group during gestation days 7-14. No significant effects on gestational body 

weight were observed in the low-dose group. Body weight gain during lactation was not 

different from controls at any dose level. (No body weight change calculations were provided 

for males; also, body weight changes for females during premating and mating periods were not 

reported.)

A treatment-related effect on food consumption was evident in all groups of treated males and 

females in comparison to controls, although these reductions were not dose-related. Food 

consumption was significantly lower than controls during Weeks 1-2 (premating period) for all 

groups of treated males, during Weeks 5-7 (postmating period) for mid-dose males, and during 

Weeks 6-7 for low-dose males. Food consumption during premating, gestation, and lactation 

was significantly lower than controls in all treated female groups, except the low-dose group 

during lactation. 

No test article-related organ weight changes were noted in rats of either sex. Test article-related 

macroscopic changes were noted in female rats, but not in males. Black or red foci were 

observed in the glandular stomach of 3/8 of the mid-dose and 4/7 of the high-dose females that 

died prior to scheduled sacrifice. Histopathological lesions observed in high-dose animals also 

included minimal focal necrosis and moderate periportal vacuolation of hepatocytes in one 

female, edema of the stomach in two of seven animals that died, and erosion/ulcer in the 

glandular stomach in one female that died. Test article-related microscopic changes were noted 
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in the liver and glandular stomach of female rats at the high dose, but not in males at any dose 

level. 

Discolored urine (brown, orange, or red) was observed in all males and females in the treated 

groups at one or more daily intervals. In addition, decreased activity was observed in four and 

five females in the mid- and high-dose groups, respectively, and one male in the low-dose group. 

The mating, fertility, and fecundity indices for treated males and females (83.3-100%) were 

similar to controls (91.7-100.0%). Likewise, the copulatory interval (days to mating) was 

unaffected by treatment and ranged from 2.4 to 3.1 days in the treated groups in comparison to 

3.0 days in the controls. All pregnant animals in the control and low-dose groups delivered and 

retained litters through lactation day 4. In the mid-dose group, 10 females delivered, but only 

two females retained litters through lactation day 4. In the high-dose group, four females 

delivered, but only one retained a litter through lactation day 4.

Treatment produced a lengthening of the estrous cycle; mean estrous cycle length (number of 

days per cycle) was increased in treated females (4.7, 5.6, and 4.8 days in the low-, mid-, and 

high-dose groups, respectively) in comparison to controls (4.4 days), but the difference was only 

statistically significant in the mid-dose group. The number of estrous cycles per animal over the 

2-week evaluation period was significantly decreased in all treated groups (2.5, 2.2, and 

2.7 cycles per animal in the low-, mid-, and high-dose groups, respectively) in comparison to 

controls (3.6 cycles per animal). The total number of implantation scars per litter was 

significantly reduced at the mid- and high dose (13.0 and 12.5, respectively) in comparison to 

controls (15.5 scars). Additionally, mean post-implantation loss per litter was significantly 

higher (31.09%) at the high dose compared to controls (6.07%). 

The number of pups delivered (live + dead) per litter was significantly decreased in the mid- and 

high-dose groups. The number of liveborn pups per litter in these groups was decreased with an 

increase in stillborn pups. At the low dose, no effect of treatment was evident from parturition 

data, litter size, or pup survival to lactation day 4; the total number of pups born, as well as the 

numbers of live and stillborn pups, were similar to controls. Pup sex ratio (% males per litter) in 

the treated groups was considered comparable to controls and unaffected by treatment. Pup 

survival to lactation day 4 (per litter) in the low-dose group (89%) was not significantly different 

from the controls (97%). Pup survival to lactation day 4 for the mid- and high-dose groups (16.5 

and 33.3%, respectively) was significantly lower than that of the control. Very few pups 

survived to lactation day 4 in the high-dose group, but no clinical observations were noted for 

these pups. 

Mean pup body weights (by sex and sexes combined) in the low-dose group were similar to 

control weights at birth, but 8-10% lower than controls by lactation day 4; however, pup weights 

at the low dose were not significantly different from those in the control group on either lactation 

day 0 or 4. In the mid-dose group, pup weights were significantly lower (22-25%) in comparison 

to controls on lactation day 0. Only two litters remained on lactation day 4 for the mid-dose 

group and, although statistical analysis could not be conducted due to the small litter size, 

weights were 51-58% lower than controls. Due to the small number of litters in the high-dose 

group, statistical analysis was not conducted for pup body weights for either time point, but 
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weights on lactation day 0 were 2-7% lower than controls and weights on lactation day 4 were 

10-11% lower than controls. The lower pup weights were considered indicative of a treatmentrelated response. No macroscopic observations were evident in any pups (stillborn, died on 

study, or sacrificed on lactation day 4) at necropsy. 

CONCLUSIONS

Considerable mortality was seen in females at the mid- and high-dose levels. Decreased body 

weights were observed in females at all dose levels and in males at the mid- and high dose. 

Reduced food consumption was observed in males and females at all dose levels. Therefore, the 

LOAEL for systemic toxicity was a nominal dose of 150 mg/kg-bw/day (88 mg/kg-bw/day 

calculated from measured concentrations); a NOAEL for systemic toxicity was not established. 

Mating, fertility, and fecundity indices (male and female) in the treatment groups were 

comparable to controls. The number of estrous cycles per animal was significantly decreased in 

all treated groups in comparison to controls. Estrous cycle length was increased at the mid-dose. 

Effects seen at both the mid- and high dose included a decrease in pups born, fewer liveborn

pups, an increase in stillborn pups, and a decrease in implantation scars per litter. Also, pup 

survival was poor in the mid- and high-dose groups with only two and one litters, respectively, 

surviving to lactation day 4. Pup body weights were significantly reduced at the mid-dose level 

at lactation day 0; combined male and female pup weights were reduced by 21.7% compared 

with controls. Mean pup body weights (combined male and female) were also decreased by 

57.5% at the mid-dose level at lactation day 4, 4.2% at the high-dose on lactation day 0, and 11% 

at the high-dose on lactation day 4, but statistical comparisons with control values could not be 

conducted because the sample sizes consisted of only 1-2 litters. An increase in postimplantation loss per litter was observed at the high dose. The LOAEL for reproductive toxicity 

was a nominal dose of 150 mg/kg-bw/day (88 mg/kg-bw/day calculated from measured 

concentrations) based on a decrease in the number of estrous cycles per animal; a reproductive 

NOAEL was not established. The LOAEL for developmental toxicity was a nominal dose of 

600 mg/kg-bw/day (405 mg/kg-bw/day calculated from measured concentrations) based on an 

increase in the number of stillborn pups and decreases in the total number of pups delivered, 

number of liveborn pups, number of implantation scars, pup body weight on lactation days 0 and 

4, and pup survival to lactation day 4; the NOAEL for developmental toxicity was a nominal 

dose of 150 mg/kg-bw/day (88 mg/kg-bw/day calculated from measured concentrations).

STUDY RELIABILITY

[2] Reliable with restrictions. Deficiencies include: (1) dosing formulations were not 

homogeneous mixtures; (2) variation in dose levels occurred during the study; (3) numbers of 

corpora lutea were not measured; (4) pre-implantation losses were not reported; (5) the study 

report was missing calculations of body weight change for males (all intervals) and for females 

during pre-mating and mating periods; (6) the results/discussion section of the study report does 

not explicitly state whether effects on pup body weights are expressed as means calculated and 

statistically analyzed on a per-pup or a per-litter basis. 
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REFERENCES

MPI Research, Inc. 2012. An Oral Reproduction/Developmental Toxicity Screening Study of 

Anthraquinone (CAS #84-65-1) in Rats. Study No. 1271-001. Date of Study Completion: 

February 6, 2007. Revised Report Dated May 4, 2012. Available at: 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2005-0033-0413 as of 

November 8, 2012.
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D. SUPPLEMENTAL DATA (NON-SIDS ENDPOINTS)

D-1. Skin Irritation

Study 1: Dermal irritation study in rabbits (From ECHA website, Exp Key Skin 

irritation/corrosion.001)

Title: Not provided

TEST SUBSTANCE 

Identity: 9,10-Anthracenedione, CASRN 84-65-1 (termed 9,10-Anthraquinone in the ECHA 

summary)

Remarks: Purity: 98.9 wt % (main component) other significant impurities include anthracene 

0.01 wt %, 2,3-Naphthalic anhydride 0.25%, Naphtho[2,3-b]thiphenquinone 0.33%, 

1,4-Anthraquinone 0.32%; Batch No. V 1161; Appearance: yellow crystalline powder

METHOD

Method/guideline followed: EU Method B.4 (Acute Toxicity: Dermal Irritation/Corrosion), 

Council Regulation (EC) No.440/2008, published in O.J. L 142, 2008.

Test type: Acute Dermal Irritation/Corrosion (in vivo)

GLP compliant?: Yes

Year study performed: 2009

Test species: New Zealand White rabbits

Number of animals/sex/dose: Three animals total, sex not indicated

Route of administration: Dermal

Vehicle: Unchanged (no vehicle)

Doses/concentrations tested: 0.5 g

Statistical methods used: None

Test method/conditions remarks: Three rabbits were exposed to 0.5 g of test substance, 

applied onto clipped skin for 4 hours using a semi-occlusive dressing. At first, the test substance 

was applied on the skin of one rabbit (test animal No. 7). The animal was investigated 

3 minutes, 1 hour, and 4 hours after application of the test substance, and following no signs of 

irritation, two additional rabbits (rabbit No. 8 and No. 9) were dosed in similar manner for a 
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4-hour exposure period. Skin reactions were evaluated after patch removal and at 1, 24, 48, and 

72 hours after exposure. 

Test Animals

- Source: breeding farm VELAZ s.r.o., Koleč u Kladna, Czech Republic, RČH CZ 21760152

- Age at study initiation: 4-5 months

- Weight at study initiation: 2.4-3.1 kg 

- Housing: individually in cages without bedding in conventional animal room

- Diet: pelleted standard diet TM-MAK 1 for rabbits and guinea-pigs ad libitum

- Water: drinking tap water ad libitum

- Acclimation period: 5 days

Environmental Conditions

- Temperature (°C): room temperature 17-23°C, permanently monitored 

- Humidity (%): 30-70%, permanently monitored

- Air changes (per hour): approximately 15 air changes per hour

- Photoperiod (hours dark / hours light): 12 hour light/dark cycle 

RESULTS

Endpoint value(s): Not irritating; mean erythema and edema scores at 24, 48, and 72 hours 

were 0. 

Results remarks: No evidence of a corrosive effect or symptoms of irritation were observed on 

the skin of the rabbits tested. No skin irritation was caused by 4-hour exposure to the test 

substance. No other signs of intoxication were observed. Based on these results and according 

to the EC criteria for classification and labeling requirements for dangerous substances and 

preparations (Council Directive 67/548/EEC, Annex VI, part 3.2.6.1), the test substance does not 

have to be classified for skin irritation and has no obligatory labeling requirement for skin 

irritation. 

CONCLUSIONS

The test substance was not irritating to rabbit skin.

STUDY RELIABILITY

[1] Reliable without restrictions. This study was carried out in accordance with internationally 

valid GLP principles. The summary did not provide a full citation for the study report.

REFERENCES

Study available from ECHA website, http://echa.europa.eu, first row of the “Search results” table 

resulting from a “Chemical Substance Search” for CASRN 84-65-1. Study indicated as Exp Key 

Skin Irritation/corrosion.001. The data source is referred to as a “study report”, with a report 

date of 2009-11-08. No other reference information is provided.
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Copyright Notice: © European Chemicals Agency, 2007-2011. Reproduction is authorised 

provided the source is fully acknowledged in the form "Source: European Chemicals Agency, 

http://echa.europa.eu/", and provided written notification is given to the ECHA Communication 

Unit (info (at) echa.europa.eu).
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Study 2: Dermal irritation study (From ECHA website, Exp Key Skin irritation/

corrosion.002)

Title: Not provided

TEST SUBSTANCE 

Identity: 9,10-Anthracenedione, CASRN 84-65-1 (termed 9,10-Anthraquinone in the ECHA 

summary)

Remarks: Purity: 98.9 wt. % (main component) other significant impurities include anthracene 

0.01 wt%, 2,3-Naphthalic anhydride 0.25%, Naphtho[2,3-b]thiphenquinone 0.33%, 

1,4-Anthraquinone 0.32%; Batch No. V 1161; Appearance: yellow crystalline powder. 

METHOD

Method/guideline followed: Draft OECD and EC Test Guidelines and Standard Operating 

Procedure for EpiDermMTM published by ZEBET

Test type: Human epidermal model EpiDermTM Test (in vitro)

GLP compliant?: Yes

Year study performed: 2009

Test species: EpiDermTM tissues

Number of animals/sex/dose: N/A

Route of administration: Dermal

Vehicle: 25 μL sterile PBS (negative control)

Doses/concentrations tested: 25 mg

Statistical methods used: None specified

Test method/conditions remarks: The test substance (25 mg) was placed directly, without 

grinding, atop the tissue previously moistened with 25 μL of sterile PBS for 60 minutes. Nine 

tissues were used for the experiment: three per test substance, three for positive control (5% 

SDS), and three for negative control (PBS). After rinsing, tissues were postincubated for 

42 hours, incubated for 3 hours with MTT, and extracted for 2 hours with shaking. OD570 of 

isopropanol extracts was measured on a spectrophotometer. Relative cell viability was 

calculated for each tissue as the percent of the mean viability of the negative control tissues. 
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Test System: The reconstructed human epidermal model EpiDermTM (EPI-200, MatTek, 

Ashland, USA) consists of normal human-derived epidermal keratinocytes, which have been 

cultured to form a multilayered highly differentiated model of the human epidermis. The 

EpiDermTM System is manufactured according to defined quality assurance procedures. The 

EpiDermTM tissues (surface 0.63 cm2) are cultured on specially prepared cell culture inserts and 

shipped world-wide as kits, containing 24 tissues on shipping agarose together with the 

necessary amount of culture media.

Criteria used for interpretation of results: EU

Deviations: 24 hours preincubation period was omitted due to time shortage – deviation from 

the MatTek instruction

RESULTS

Endpoint value(s): Non-irritating

Results remarks: Following a 60-minute incubation period of the test substance with MTT 

medium, no changes in coloring were observed compared to the concurrent control without the 

test substance. The test substance did not reduce MTT directly. The mean OD570 value for the 

test substance was 1.597 at 60 minutes. 

Average viability of affected tissues is 91.3% of negative control average value (i.e., viability is 

> 50%). When this value is compared with classification criteria of the method (chapter 3.8), the 

substance could be classified as non-irritant (NI).

All assay acceptance criteria were met.

Table 1: OD570 values obtained at the MTT test, their averages, standard deviations (%), and 

relative viabilities

10-12/06 2009 1 2 3 Mean SD %NC

NS PBS 1.833 1.479 1.935 1.749 0.195 100.0

% NC 104.803 84.563 110.635 100.0 11.173

TS Anthraquinone 1.973 1.238 1.580 1.597 0.300 91.3

% NC 112.807 70.783 90.337 91.309 17.170 NI

PC 5% SDS 0.115 0.123 0.099 0.112 0.010 6.4

% NC 6.575 7.033 5.660 6.423 0.570 R38

NC negative control 

PC positive control 

TS test substance 

mean arithmetic mean 

% NC viability of single tissues compared with negative control 

SD standard deviation calculated from individual % tissue viabilities 
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CONCLUSIONS

Under the above-described experimental design, the test substance was nonirritating for 

EpiDermTM tissues. 

STUDY RELIABILITY

[2] Reliable with restrictions. The summary did not provide a full citation for the study report, 

provide a full description of the test methodology and its principles, define several of the 

acronyms presented in the table of study results, or present a clear interpretation of study results. 

According to the MatTek instruction, a deviation occurred during this study when the 24-hour 

preincubation period was omitted due to a time shortage.

REFERENCES

Study available from ECHA website, http://echa.europa.eu, first row of the “Search results” table 

resulting from a “Chemical Substance Search” for CASRN 84-65-1. Study indicated as Exp Key 

Skin Irritation/corrosion.002. The data source is referred to as a “study report”, with a report 

date of 2009-06-29. No other reference information is provided.

Copyright Notice: © European Chemicals Agency, 2007-2011. Reproduction is authorised 

provided the source is fully acknowledged in the form "Source: European Chemicals Agency, 

http://echa.europa.eu/", and provided written notification is given to the ECHA Communication 

Unit (info (at) echa.europa.eu).
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D-2. Eye Irritation

Study 1: Eye irritation study in rabbits (From ECHA website, Exp Key Eye Irritation.001)

Title: Not provided

TEST SUBSTANCE 

Identity: 9,10-Anthracenedione, CASRN 84-65-1 (termed 9,10-Anthraquinone in the ECHA 

summary)

Remarks: Purity: 98.9 wt. % (main component) other significant impurities include anthracene 

0.01 wt%, 2,3-Naphthalic anhydride 0.25%, Naphtho[2,3-b]thiphenquinone 0.33%, 

1,4-Anthraquinone 0.32%; Batch No. V 1161; Appearance: yellow crystalline powder 

METHOD

Method/guideline followed: EU Method B.5 (Acute Toxicity: Eye Irritation/Corrosion), 

Council Regulation (EC) No.440/2008, published in O.J. L 142, 2008. 

Test type: Acute Eye Irritation

GLP compliant?: Yes

Year study performed: 2009

Test species: New Zealand White Rabbits

Number of animals/sex/dose: 3 males

Route of administration: Ocular

Vehicle: Unchanged (no vehicle)

Doses/concentrations tested: 0.1 g

Statistical methods used: None specified

Test method/conditions remarks: The test substance was initially applied to the eye of a single 

animal for 24 hours, and upon observation of no corrosive effects, two additional animals were 

dosed in a similar manner. Following exposure, observations were made at 1, 24, 48, and 

72 hours after application and then once daily. Eyes were not rinsed following instillation.

Tool used to assess score: fluorescein and ophthalmoscopy
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Test animals

- Source: breeding farm VELAZ s.r.o., Koleč u Kladna, Czech Republic, RČH CZ 21760152

- Age at study initiation: 4-5 months

- Weight at study initiation: 2.5-3.0 kg

- Housing: Conventional animal room – individually in metallic cages

- Diet: Standard pelleted diet TM-MAK 1 for rabbits and guinea pigs ad libitum

- Water: Drinking tap water ad libitum

- Acclimation period: 5 days

Environmental Conditions

- Temperature (°C): Room temperature: 20 ± 3°C, permanently monitored 

- Humidity (%): Relative humidity: 30-70%, permanently monitored

- Air changes (per hour): approximately 15 air changes per hour

- Photoperiod (hours dark/hours light): Light period: 12 hour light/12 hour dark

RESULTS

Endpoint value(s): Not irritating

Results remarks: The mean irritation scores for cornea, iris, conjunctivae, and chemosis at 24, 

48, and 72 hours were 0, 0, 1, and 0, respectively. Effects were detected in all three animals at 

1 hour after application, and in two animals effects persisted up to 24 hours after application. 

Changes at 1 hour after application included: strong lacrimation, redness of conjunctivae –

diffuse, crimson color, individual vessels not easily discernible; chemosis – some swelling above 

normal. 

Changes at 24 hours after application included: conjunctivae – some blood vessels hyperemic 

(injected). 

Changes at 48 and 72 hours after application: no alterations. 

No clinical signs of systemic intoxication were detected. Individual results for each animal 

tested are provided below.

Rabbit No. 10:

strong lacrimation was observed 1 hour after application

1 hour: Conjunctivae – some blood vessels hyperaemic (injected)

Chemosis – no alterations

Cornea – no alterations 

Iris – no alterations

24 hours: No alterations

48 hours: No alterations

72 hours: No alterations

Rabbit No. 11:

1 hour: Conjunctivae – diffuse, crimson color, individual vessels not easily discernible

Chemosis – no alterations

Cornea – no alterations 
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Iris – no alterations 

24 hours: Conjunctivae – some blood vessels hyperaemic (injected)

48 hours: No alterations

72 hours: No alterations

Rabbit No. 12: 

1 hour: Conjunctivae – diffuse, crimson color, individual vessels not easily discernible 

Chemosis – some swelling above normal Lacrimation

24 hours: Conjunctivae – some blood vessels hyperaemic (injected)

48 hours: No alterations

72 hours: No alterations

CONCLUSIONS

Examination of eye irritation after single application demonstrated that the test substance is not 

irritating to the eyes of rabbits. 

STUDY RELIABILITY

[1] Reliable without restrictions. This study was carried out in accordance with internationally 

valid GLP principles. The summary did not provide a full citation for the study report.

REFERENCES

Study available from ECHA website, http://echa.europa.eu, first row of the “Search results” table 

resulting from a “Chemical Substance Search” for CASRN 84-65-1. Study indicated as Exp Key 

Eye Irritation.001. The data source is referred to as a “study report”, with a report date of 2009-

11-08. No other reference information is provided.

Copyright Notice: © European Chemicals Agency, 2007-2011. Reproduction is authorised 

provided the source is fully acknowledged in the form "Source: European Chemicals Agency, 

http://echa.europa.eu/", and provided written notification is given to the ECHA Communication 

Unit (info (at) echa.europa.eu).
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Study 2: Eye irritation study in rabbits (From ECHA website, Exp Key Eye Irritation.002)

Title: Not provided

TEST SUBSTANCE 

Identity: 9,10-Anthracenedione, CASRN 84-65-1 (termed 9,10-Anthraquinone in the ECHA 

summary)

Remarks: Purity: 98.9 wt. % (main component) other significant impurities include anthracene 

0.01 wt %, 2,3-Naphthalic anhydride 0.25%, Naphtho[2,3-b]thiphenquinone 0.33%, 

1,4-Anthraquinone 0.32%; Batch No. V 1161; Appearance: yellow crystalline powder 

METHOD

Method/guideline followed: MatTek Ocular Irritation Protocol: Neat Method (MTT ET-50), 

Rev. 1/1/01.

Test type: In vitro Eye irritation in human corneal model EpiOcular TM Ocular Irritation

GLP compliant?: Yes

Year study performed: 2009

Test species: Human corneal model EpiOcular TM

Number of animals/sex/dose: N/A 

Route of administration: In vitro

Vehicle: Water

Doses/concentrations tested: 100 mg 

Statistical methods used: None specified 

Test method/conditions remarks: The test substance was mixed with water (1:2) and applied 

as paste using the applicator pin. At application, it was assured that the tissues were completely 

covered by the paste. Tissues were exposed for 3, 30, and 60 minutes. Three tissues were used 

for each time interval: three for positive control (PC) and three for negative control (NC). After 

rinsing and soaking in medium, tissues were incubated with MTT for 3 hours and then extracted 

overnight without shaking. OD570 of isopropanol extracts was measured on a spectrophotometer. 

Relative cell viability was calculated for each of the three tissues as a percent of the mean 

viability of the negative control. From a semi-log scale - the percent viability (linear y axis) 

versus the dosing time (log x axis), the ET50 value was calculated from slope of the line obtained. 
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RESULTS

Endpoint value(s): Non-irritating

Results remarks: The mean OD570 values at 3, 30, and 60 minutes were 1.732, 1.573, and 1.73. 

Following a 60-minute incubation period of the test substance with MTT medium, no changes in 

coloring were observed compared to the concurrent control without the test substance. The test 

substance did not reduce MTT directly

Table 1: OD570 values obtained in the MTT test, their averages, standard deviations (%), and 

relative viabilities

OD570 Mean SD Tolerable interval Viability

Chemical 1 2 3 of values (% NC)

Water for inj..60 min 1.756 1.741 1.779 1.759 0.016 1.495 2.022 100.0

Test substance, 3 min 1.685 1.727 1.784 1.732 0.041 1.472 1.992 98.5

Test substance, 30 min 1.473 1.674 1.572 1.573 0.082 1.337 1.809 89.4

Test substance, 60 min 1.661 1.774 1.754 1.730 0.049 1.470 1.989 98.4

0.3% Triton X-100 15 min 1.089 1.105 1.137 1.110 0.020 0.944 1.277 63.1

0.3% Triton X-100 45 min 0.337 0.296 0.267 0.300 0.029 0.255 0.345 17.1

As it is possible to see from the results given above, average viabilities of affected tissues were 

98.5% after 3 minutes of treatment, 89.4% after 30 minutes, and 98.4% after 60 minutes of 

administration. Viability did not decrease with time. The ET50 value calculated was high –

3.86*1020 minutes, which is higher than 60 minutes, showing that the test substance did not 

affect the viability of the tissue and therefore, did not have an irritation effect.

All criteria of assay acceptance have been met: OD570 of negative control - 1.719 – lies in 

interval 1.0 ≤ 1.759 ≥ 2.5, ET50 for 0.3% Triton X-100 lies between 15 and 45 minutes

(20.52 minutes) and all OD570 values fulfill the condition average ± 15%. 

CONCLUSIONS

In the experiment arrangement given above, the test substance was nonirritating in EpiOcularTM 

model.

STUDY RELIABILITY

[2] Reliable with restrictions. This study was carried out in accordance with internationally valid 

GLP principles. The summary did not provide a full citation for the study report, provide a full 

description of the test methodology and its principles, or present a clear interpretation of study 

results. 
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REFERENCES

Study available from ECHA website, http://echa.europa.eu, first row of the “Search results” table 

resulting from a “Chemical Substance Search” for CASRN 84-65-1. Study indicated as Exp Key 

Eye Irritation.002. The data source is referred to as a “study report”, with a report date of 2009-

09-06. No other reference information is provided.

Copyright Notice: © European Chemicals Agency, 2007-2011. Reproduction is authorised 

provided the source is fully acknowledged in the form "Source: European Chemicals Agency, 

http://echa.europa.eu/", and provided written notification is given to the ECHA Communication 

Unit (info (at) echa.europa.eu).
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D-3. Skin Sensitization

Study 1: Dermal sensitization study in mice (From ECHA website, Exp Key Skin 

Sensitization.001)

Title: Not provided

TEST SUBSTANCE

Identity: 9,10-Anthracenedione, CASRN 84-65-1 (termed 9,10-Anthraquinone in the ECHA 

summary)

Remarks: Purity: 98.9 wt. % (main component) other significant impurities include anthracene 

0.01 wt %, 2,3-Naphthalic anhydride 0.25%, Naphtho[2,3-b]thiphenquinone 0.33%, 

1,4-Anthraquinone 0.32%; Batch No. V 1161; Appearance: yellow crystalline powder 

METHOD

Method/guideline followed: EU Method B.42 (Skin Sensitization: Local Lymph Node Assay), 

Council Regulation (EC) No. 440/2008, published in O.J. L142, 2008. 

Test type: Skin sensitization

GLP compliant?: Yes

Year study performed: 2009

Test species: Balb/c mice, female

Number of animals/sex/dose: Five test animals

Route of administration: Dermal

Vehicle: DAE 433 - mixture of 40% dimethylacetamide, 30% acetone, and 30% ethanol 

Doses/concentrations tested: Test substance: 30%, 3%, 0.3% (w/v) in DAE 433; Positive 

control: 0.5% (w/v) DNCB (dinitrochlorobenzene). 

Test method/conditions remarks: Five mice per group were exposed to test and control 

substances on the dorsum of both ears (25 μL each) once a day during 3 consecutive days. The 

application was performed very slowly by micropipette. The application form of test substance 

was prepared immediately before administration.

Primary proliferation of lymphocytes in the lymph node draining the site of application was 

evaluated based on radioactive labeling. The ratio of proliferation in treated groups to that in 
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vehicular controls, termed the Stimulation Index (SI), was determined. Statistical evaluation of 

ear weight was performed for elimination of false positive findings with certain skin irritants. 

Test Animals

- Source: Breeding farm VELAZ s.r.o., Koleč u Kladna, Czech Republic, RČH CZ 21760118

- Age at study initiation: 8-10 weeks (at start of dosing)

- Weight at study initiation (range): 21.1-24.0 g (at start of dosing)

- Animal caging: Animals in groups of maximum six in macrolon cages with sterilized softwood 

shavings.

- Diet: Pelleted standard diet for experimental animals ad libitum.

- Water: Drinking tap water ad libitum

- Acclimation period: At least 5 days

Environmental Conditions

- Temperature (°C): Room temperature 22 ± 3°C, permanently monitored

- Humidity (%): Relative humidity 30-70%, permanently monitored

- Air changes (per hour): approximately 15 air changes per hour

- Photoperiod (hours dark / hours light): 12 hours light/dark cycle: 6am-6pm/6pm-6am

Range-finding tests

The highest concentration, 30% (maximum technically practicable concentration), was 

administered to three animals to assess a possible systemic toxicity. The route of administration 

was same as in the main study. During the pilot experiment, no clinical symptoms of systemic 

toxicity and no macroscopic changes (after necropsy) were found in any of the three animals.

Main Study

Animal Assignment and Treatment

Animals were subjected to a clinical examination (health check) shortly after arrival. Study 

animals were randomly allocated to the dose groups manually and assigned animal numbers.

Evaluation of Results

Cell proliferation: When the SI for any single treatment dose group is ≥ 3 (exceeded threshold) 

and clear concentration dependence is recorded, the test substance is regarded as a skin 

sensitizer.

Ear weight – irritation effect: When a statistically significant increase in ear weight after 

treatment by the test substance together with clear concentration dependence of the effect is 

recorded, the endpoint is considered positive – test substance may cause the irritation of skin. 

Treatment Preparation and Administration

Dosage volume: 25 μL/ear/animal

Preparation for administration: All suspensions were prepared by suspending an appropriate 

amount of the test substance in the vehicle to obtain a concentration of 30, 3, or 0.3% (w/v). The 

suspensions were prepared before the start of application by mixing on magnetic stirrer; mixing 

continued during application.
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Statistics: For statistical calculations, the software Statgraphic® Centurion (version XV, USA) 

was used. At first, the global comparison of all three values of the concentration groups with 

vehicle control was performed by applying the non-parametric Kruskal-Wallis test, and then the 

non-parametric two-group Mann-Whitney rank test (probability level 0.05) was applied to all 

two-group comparisons.

RESULTS

Endpoint value(s): Sensitizing 

Results remarks: The animals exposed to the test substance at the lowest and highest dose 

levels showed no pathological skin reactions and no other negative clinical symptoms of 

intoxication throughout the experiment. Slight hyperemia of skin and swelling of ears was 

recorded at the highest dose level. There was no significant difference in body weight increment 

of any group in comparison to the vehicle control. 

The SI for the test groups treated by the test substance was increased with dependence on the 

dose level. In the highest dose level, the SI was 6.53 (exceeded threshold), and the SI for the 

middle and lowest dose groups was 1.80 and 1.60, respectively, which were both below 

threshold. 

Summary of results

Group Radioisotope incorporation Ear weight

Mean DPM SI Mean (mg)

NC 1026.2 1.00 22.84

PC 10738.8 10.46+ 27.50*

30% 6705.4 6.53+ 28.22*

3% 1845.6 1.80 24.84*

0.3% 1637.4 1.60 23.80*

Figures with asterisk = values statistically significant on probability level 0.05 (Mann-Whitney 

test) 

Figures with cross = values ≥ 3 

NC – Negative control group 

PC – Positive control group 

DPM – Disintegrations per minute

The test substance also showed a tendency to increase ear weight with concentrationdependence. Ear weight of animals at all dose levels was statistically significantly increased. 

The result of skin irritation effect was considered positive, meaning that skin irritation was 

observed. 

The positive control substance DNCB elicited a reaction pattern comprised of a statistically 

significant increase in ear weight and a Stimulation Index of cell proliferation of 10.5, which was 

in congruence with the expected mode of action as a contact allergen. 

Based on these results, the test substance had a positive result in this LLNA test.
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CONCLUSIONS

The test substance gave a positive result in the LLNA test. Positive results in cell proliferation, 

with no clinical symptoms of systemic toxicity, revealed that the test substance could be a 

contact allergen in mice but the potential irritation effect does not rule out the possibility that it 

could be a false positive result.

STUDY RELIABILITY

[1] Reliable without restrictions. This study was carried out in accordance with internationally 

valid GLP principles. The summary did not provide a full citation for the study report and did 

not identify the radioactive substance that was used to evaluate proliferation of lymphocytes in 

lymph nodes that drain the ear.

REFERENCES

Study available from ECHA website, http://echa.europa.eu, first row of the “Search results” table 

resulting from a “Chemical Substance Search” for CASRN 84-65-1. Study indicated as Exp Key 

Skin Sensitization.001. The data source is referred to as a “study report”, with a report date of 

2009-07-29. No other reference information is provided.

Copyright Notice: © European Chemicals Agency, 2007-2011. Reproduction is authorised 

provided the source is fully acknowledged in the form "Source: European Chemicals Agency, 

http://echa.europa.eu/", and provided written notification is given to the ECHA Communication 

Unit (info (at) echa.europa.eu).
Draft HPV1/HPV2 Chemical Data Adequacy Review (May 2013) CASRN 84-65-1

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D-4. Genetic Toxicity – Other

Study 1: In vivo mouse DNA damage test (Cesarone et al., 1982)

Title: Evaluation of damage to DNA after in vivo exposure to different classes of chemicals 

TEST SUBSTANCE 

Identity: 9,10-Anthracenedione, CASRN 84-65-1 (referred to as Anthraquinone in the study 

description) 

Remarks: None specified

METHOD

Method/guideline followed: No data

Test type: Induction of single-strand DNA breaks in liver and kidney tissue

GLP compliant?: No data

Year study performed: 1982 (date of published journal article)

Test species: Swiss CD-1 male mice

Number of animals/sex/dose: No data

Route of administration: Intraperitoneal 

Vehicle: No data

Exposure duration: 4 hours

Doses tested: 0 (solvent control) and 250 mg/kg-bw 

Statistical methods used: None specified

Test method/conditions remarks: The test substance was tested for its capability of inducing 

single-stranded DNA-breaks in the liver or kidney of Swiss CD1 male mice. DNA damage was 

evaluated by the alkaline elution technique coupled with a microfluorometric method for DNA 

assay. This technique involves an in vivo treatment of rodents with a given compound, isolation 

of nuclei from target tissues at 4 hours after test substance administration, and lysis on a 

membrane filter. DNA is eluted, using an alkaline buffer, as a function of the molecular weight. 
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RESULTS

Genotoxicity result: Positive

Cytotoxic dose: No data

Results remarks: Increased elution rates were obtained for both liver and kidney cells.

CONCLUSIONS

The test chemical appears to have the potential to induce DNA strand breaks in liver and kidney 

cells of mice (in vivo).

STUDY RELIABILITY

[4] Not assignable. This was not a guideline study, test substance purity and composition were 

not stated, and limited study details were provided. 

REFERENCES

Cesarone, C.F., Bolognesi, C., and Santi, L. 1982. Evaluation of damage to DNA after in vivo 

exposure to different classes of chemicals. Arch. Toxicol., Suppl. 5:355-359. 
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E. DATA ADEQUACY SUMMARY TABLE 

Endpoint Study Information 

Available

Acceptable Data Gap

Aquatic Effects

Acute Toxicity to Fish Yes Yes No

Acute Toxicity to Aquatic Invertebrates Yes Yes No

Toxicity to Plants (Algae) Yes No No1

Health Effects

Acute Toxicity Yes Yes No

Repeated-Dose Toxicity Yes Yes No

Gene Mutation Yes Yes No

Chromosomal Aberrations Yes Yes No

Reproductive Toxicity Yes Yes No

Developmental Toxicity Yes Yes No

1 Although no adequate algal toxicity studies are available, the toxicity to aquatic plants endpoint 

is not considered to be a data gap at this time, given that: (1) low toxicity has been observed in 

fish and daphnids (i.e., no adverse effects at or near the limit of water solubility); and (2) 

according to EPA’s 1987 test rule for CASRN 84-65-1 (Anthraquinone; Final Reporting and 

Recordkeeping Requirements and Test Rule; 52 FR 21018, June 4, 1987), “sufficient data are 

available from the study done by Chillingworth [1974]... to reasonably predict anthraquinone’s 

toxicity to algae”. There is no need for further testing of the toxicity to aquatic plants endpoint at 

this time.