SRC TR

- 13

- 016

HPV 1 /HPV2 TEST RULE DATA ADEQU ACY 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. 00 4

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 , 201 3Draft HPV 1/HPV2 Chemical Data Adequacy Review (May 2013) CASRN 84

- 65

- 1

i 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. Repeat ed

- 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

................................ ................................ ......... 120Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

1 A. SOURCE MATERIAL USED FOR ASSESSMENT

In this document, toxicological data for 9,10

- a nthracenedione (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.”

I n 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 (Te sting 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

- 04 e1 ) in t he 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; ht tp://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) revisio n 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 un reliable 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) w ebsite ( 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.Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

2 ( 3 ) Chemical Pr oducts 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 t hese data are not considered adequate to satisfy the C6 testing re quirement, 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 li mit 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 Chillingwo rth [1974]... to reasonably predict anthraquinone’s toxicity to algae”. Therefore, t here is no need for further testing of the toxicity to aquatic plants endpoint at this time.

T his 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 endpoint s , 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 Hig h 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 o f aminoanthraquinone dyes to fish and algae. M.S.P.H. Thesis, Dept. Environ. Sci. Eng., Univ. North Carolina, Chapel Hill.Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

3 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 pag e 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 su mmary document.Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

4 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 h ours

Concentrations tested : No data

Test method/conditions remarks : No data

RESULTS

Endpoint value(s) : 96

- hour LC 50 > 190 μ g/L; NOEC > 190 μ g/L

Results remarks : T he Office of Pesticide Programs “ Anthraquinone (122701) Fact Sheet ” reports the LC 50 and NOEC in units of “Fg/L”, i.e., LC 50 > 190 F g/L and NOEC > 190 F g/L

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

- 65

- 1

5 84

- 65

- 1, the units are assumed to be μg/L; the use of “Fg/L” rath er 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 LC 50 for Lepomis macrochirus was > 190 μ g/L.

STUDY RELIABILITY

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

REFERENCE S

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

- Dec

98.pdf as of May 22 , 2013

.Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

6 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 t rout ( Oncorhynchus mykiss )

Analytical monitoring : No data

Exposure duration : 96 h ours

Concentrations tested : No data

Test method/conditions remarks : No data

RESULTS

Endpoint value(s) : 96

- hour LC 50 > 150 μ g/L; NOEC > 150 μ g/L

Results remarks : The Office of Pesticide Programs “Anthraquinone (122701) Fact Sheet” reports the LC 50 and NOEC in units of “Fg/L”, i.e., LC 50 > 1 5 0 Fg/L and NOEC > 1 5 0 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

.Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

7 CONCLUSIONS

The 96

- hour LC 50 for Oncorhynchus mykiss was > 150 μ g/L.

STUDY RELIABILITY

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

REFERENCE S

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

- Dec

98.pdf as of May 22, 2013.Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

8 Study 3 : Acute toxicity study in rainbow trout ( Springborn Life Sciences, Inc. , 1988 a )

Title : Acute Toxicity of Anthraquinone to Rainbow Trou t ( Salmo gairdneri ) u nder Flow

Through 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 concen trations 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 wer e 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 wa ter 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 conditio ns (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 sub lethal 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 : 1988Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

9 Test species : Rainbow t rout ( Oncorhynchus mykiss )

Analytical monitoring : Yes ; HPLC

Exposure duration : 96 hours

Concentrations tested : Nominal c oncentrations : 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 cont ained the maximum
amount of ace tone... present in any test concentration (75 μL/L). All treatmen t levels and the

c ontrols 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 pro vided 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 a nthraquinone 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 a nthraquinone under test conditions. Test conditions for this study were generally consistent with the requirements publishe d 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 a nthraquinone , under the maintained test conditions, was approximately 50 μg/L. ” [Springborn Life Sciences, Inc. 1988a , p. 14]Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

10 Details on Sampling : “ All test concentrations and the control solution were sampled and analyzed for a nthraquinone 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 a nthraquinone. Each exposure solution sample was collected from t he 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 sam ples were prepared in dilution water at a nthraquinone 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. ” [Spri ngborn 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 g rams 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 p ositioned 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 concentra tion (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. ” [Springbo rn 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 (CaCO 3 ) of 24

- 28 and 19

- 22 mg/L, respectively, and a specific conductance range of 100

- 130 micromhos per c enti m eter (μmhos/cm) (Weekly Gravity Feed Tank Water Quality Analysis Log Book). Other parameters monitored in the holding tank were a pH r ange 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 solutionsDraft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

11 (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 LC 50 > 55 μg a.i./L ( based on mean measured concentrations )

Results remarks : “ Throughout the exposure period

, a small amount of precipitate d test material was observed in the diluter system's mixing chamber

, however

, no undissolved Anthr a quinone (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, m easured concentrations of test substance varied by more than 30%
duri ng 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% fr om 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.

M easured test concentrations at nominal concentrat ions 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 EC 50 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 limi t of water solubility. ” [Springborn Life Sciences, Inc. 1988a, p. 1 7 ]Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

12 STUDY RELIABILITY

[ 3 ] Not r eliable. Th is 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) t he 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 maxi m um of 20 percent allowed by the guideline ; (2) between 0 hours and 96 hours, the measured test concentration at
the no minal concentration s of 18 and 42 μg/L varied by more than the maxi mum 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 abo ve 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 matte r of the dilution water were not re ported; ( 6 ) the water solubility determinations and adequacy of the methods to attain maximum test concentrations w ere 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 μ g/L at a temperature and pH closely approximating the fish test conditions (12 °C and pH of 7.0) ; ( 8 ) the ag e of the fish was not reported; ( 9 ) the concentration of acetone used (75 μL/L) exceed ed 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

.

REFER ENCE S

Springborn Life Sciences

, Inc . 1988 a . Acute Toxicity of Anthraquinone to Rainbow Trou t ( Salmo gairdneri ) u nder 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 Science s , Inc., 1988a.Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

13 Study 4 : Acute toxicity study in c oho salmon ( Springborn Life Sciences, Inc., 1988b )

Title : Acute Toxicity of Anthraquinone to Coho Salmon ( Orcorhynchus kisutch ) u nder Flow

Through 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 reporte d; (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 1 8 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 conce ntration 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; a nd (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 so lubility 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 : 1988Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

14 Test species : Coho salmon ( Oncorhynchus kisutch )

Analytical monitoring : Yes ; HPLC

Exposure duration : 96 hours

Concentrations tested : Nominal c oncentrations : 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 treatmen t 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 ) wi th 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 ever y 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 a nthraquinone in freshwater under the maintained test conditions. Prior to initiation of the definitive study

, preliminary testing was conducted at Spring born Life Sciences, Inc. (SLS) to establish the water solubility and acute toxicity of a nthraquinone under test conditions. Test conditions for this study were generally consistent with the requirements published in the EPA's Final Test Rule, Parts 704, 7 95

, 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 c oho
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 o xygen : 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 g rams of active ingredient) with acetone to a total volume ofDraft 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 a pproximately 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 t o deliver acetone to the solvent control aquaria. ” [Springborn Life Sciences, Inc. 1988b, p. 9

- 10]

Details on Test Organisms : “ The c oho 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

ca rbonate (CaC O 3 ) of 24

- 28 mg/L and 19

- 22 mg/L, respectively, and a specific conductance range of 100

- 130 micromhos per c enti m eter (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 ra nge 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

c oho salmon used during t his 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) g rams and total length of 59 (44

- 73) m illi m eters..

. ” [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 0.1 8 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 ini tiation and every 24 hours thereafter until the test was terminated.

T he 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 CaCO 3 instead of 20

- 35 mg/L CaCO 3 , 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 LC 50 > 45 μg a.i./L ( based on mean measured concentrations ); NOEC = 45 μg a.i./L ( based on mean measured concentrations )Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

16 Results remarks : “ Throughout the exposure period, a small amount of precipitate d test material was observed in the diluter system's mixing chamber

, however no undissolved A nthraquinone (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. 1 5]

M easur ed concentrations of test substance decreased during the 96

- hour test period. The measured concentration decreased by 47 1 , 30 1 , 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. M easured test concentrations at nominal concentrations of 18, 27, and 100 μg/L varied more than 20% between replicates at 96 hours. At 100 μ g/L (nominal)

, the difference between replicates was 21 % at 96
hours

. At 27 μ g/L (nominal) , the difference between replicates was greater than 61 % at 96
hours . At 18 μ g/L (nominal)

, the difference between replicates was greater than 3 1% at 96
hours

.

During 96 hours of exposure, no mortalities or adverse effects were observed among c oho salmon exposed to any of the tested concentrations (12

- 45 μg a.i./L).

CONCLUSIONS

The 96

- hour EC 50 for c oho 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 qual ity, solution temperature, solvent concentration, mixing period) Anthraquinone was not acutely toxic to c oho s almon at the material's limit of water solubility. ” [Springborn Life Sciences, Inc. 1988b, p. 1 7 ]

STUDY RELIABILITY

[ 3 ] Not r eliable. 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 m atter of the dilution water were not reported ; (3) the report did not indicate which pesticides were measur ed 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.Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

17 0 hours and 96 hours, the measured test concentration at the nominal concentration of 18 μg/L varied by more than the maxi mum of 30% allowed by the guideline; (9) the concentration of acetone used (75 μL/L) exceed ed 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 (1 1 ) the maximum water
solubilit y 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)

.

REFERENCE S

Springborn Life Sciences

, Inc. 1988b. Acute Toxicity of Anthraquinone to Coho Salmon ( Orcorhync hus 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 Science s , Inc., 1988b.Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

18 Study 5 : Acute toxicity study in bluegill sunfish ( Springborn Life Sciences, Inc., 1988c )

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

Through 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 po ssible 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 concentra tion 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 ho urs 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 ade quately 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 t he 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 )Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

19 Analytical monitoring : Yes ; HPLC

Exposure duration : 1 5 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 i n 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 p rovided by Durotest V ita

- Lite R

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. Prio r 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 a nthraquinone under test conditions. Test conditions for this study were generally c onsistent 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, w as 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 measure d 39 x 20 x 25 c enti m eters (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 r eplacements 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 wa s 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.Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

20 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 15 th 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 A nthraquinone

. 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 s amples 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 S olutions : “ A diluter stock solution of 1.34 mg A

. I ./mL was prepared by diluting 0.3358 g of Anthraquinone (0.3352 g rams 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 concentrat ion 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 (CaCO 3 ) of 27

- 32 mg/L and 22

- 24 mg/L, respectively, and a specific conductance range of 100

- 130 micromhos per c enti m eter (μmhos/cm) (Weekl y 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 (Week ly 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 pel leted 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) gram s and total length of 50 (42

- 59) m illi m eters (Fish Weight and Lengths Log). ” [Springborn Life Sciences, Inc. 1988c, p. 7

- 8]Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

21 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)

, i ncorrect feeding schedule (should have occurred every other day but no feeding occurred between day s 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 : 1 5

- day LC 50 > 45 μg a.i./L ( based on mean measured concentrations ); 1 5

- 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 a nthraquinone (e.g., precipitate, film on solution's surface) was observed in any of the exposure vessels during the study. ” [Springborn Life Scie nces, Inc. 1988c, p. 15

- 16 ]

M easured 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 2 7 %

.

During 1 5 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 sub stance

.

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 th is [sic] data

, i t 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 1 5

- day LC 50 for the test substance in bluegill was empirically estimated as being greater than the highest mean measured test concentration (45 μg A.I. /L). ” [Springborn Life Sciences, Inc. 1988c, p. 17 ]

STUDY RELIABILITY

[ 3 ] Not r eliable. 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 EPADraft 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 guid eline 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) exceed ed the test gui deline 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 p H 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.

REFERENCE S

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 Science s , Inc., 1988c.Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

23 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 : 198 5

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. 2 4 mg/L (mean measured)

Test method/conditions remarks : In a limit test , groups of five fathead minnow s were exposed to 0 (control) or 0.2 4 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 flow

through 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 areDraft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

24 74.6 μg/L (20°C, pH 7; Case Consulting Laboratories, 2006 1 ) and 1.35 mg/L (25 °C; Yalkowsky and Dannenfelser, 1990 2 ).

Alkalinity was 46.0 CaCO 3 /L, water hardness was 43.0 mg CaCO 3 /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 LC 100 > 0.2 4 mg/L (based on measured concentration)

Results remarks : There was no mortality in either the control or the one test concentration of
0.2 4 mg/L. Exposed fish showed no evidence of stress during the test.

CONCLUSIONS

The 96

- hour LC 100 for fathead minnow was > 0.2 4 mg/L.

STUDY RELIABILITY

[ 3 ] Not r eliable. 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.

REFERENCE S

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

- Dec

98.pdf as of May 22, 2013.

1 Case Consulting Laboratories, Inc. 2006. Physical and Chemical Characteristics of Anthraquinone : Octanol/Water Partit ion 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 Ariz ona, 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.Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

25 Study 7 : Acute toxicity study in northern squawfish, Chinook salmon, and c oho salmon (MacPhee and Ruelle , 1969)

Title : Lethal effects of 1888 chemicals upon four species of fish from wester n 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 LC 50 was not determined, only an LC 100

.

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 c oho salmon ( Oncorhynchus kisutch ). Fish ranged in size from 5 to 10 cm in length

.

Analytical monitoring : No

Exposure duration : 24 h ours

Concentrations tested : 10 mg/L

Test method/conditions remarks : The dilution water utilized for this test was collected from
Rochat Creek. O ne squawfish and one of each of the two spec ies 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 74.6Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

26 μg/L (20°C, pH 7; Case Consulting Laboratories, 2006 1 ) and 1.35 mg/L (25 °C; Ya lkowsky and Dannenfelser, 1990 2 ).

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 LC 100 ≤ 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 c oho salmon both died
between 5 an d 9 hours after test initiation

.

CONCLUSIONS

The 24

- hour LC 100 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. A n LC 50 was not determined, only a n LC 100

. Data are useful for performing a future
definitive test.

REFERENCE S

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. Phy sical 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 Databa se 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 obta ined
from EPI Suite v4.1.Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

27 Study 8 : Acute toxicity study in Mozambique tilapia ( Jaiswal et al., 1987)

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

compo unds for Oreochromis mossambicus ( Peters) Trewavas and changes induced by these
bioci des

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 LC 50 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 h ours

Concentrations tested : 13.5 mg/L

Test method/conditions remarks : Ten fish were expo sed 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 theDraft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

28 reported water solubility limit values of 74.6 μg/L (20°C, pH 7; Case Consulting Laboratories,
2006 1 ) and 1.35 mg/L (25 °C; Yalkowsky and Dannenfelser, 1990 2 ).

RESULTS

Endpoint value(s) : 48

- hour LC 100 > 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

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

CONCLUSIONS

The 48

- hour LC 100 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 n ot 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 LC 50 was not determined. The exposure duration was only 48 hours, rather than 96 hours.

REFERENCE S

Jaiswal, S.K. and Waghray

, S. 198 9

. Annotation o n 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. Novembe r 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 Sol ubility Data. CRC Press, Boca Raton, FL (ISBN 0

- 8493

- 1532

- 8). Value obtained
from EPI Suite v4.1.Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

29 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 sub stance 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 h ours

Concentrations tested : No data

Test method/conditions remarks : No data

RESULTS

Endpoint value(s) : 48

- hour L C 50 > 240 μg/L ; 48

- hour NOEC > 240 μg/L

Results remarks : T he 48

- hour L C 50 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 LC 50 and NOEC in units of “Fg/L”, i.e., LC 50 > 240 Fg/L and NOEC > 240 Fg/L. The meaning of “Fg” isDraft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

30 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 Evalua tion Review for this study were not available for review; the study report is considered to be FIFRA proprietary information.

CONCLUSIONS

The 48

- hour LC 50 for Daphnia was > 240 μg/L

.

STUDY RELIABILITY

[1] Reliable without restrictions

. This s tudy , MRID # 44496124, is considered acceptable by
OPP.

REFERENCE S

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

- Dec

98.pdf as of May 22

, 2013.Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

31 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 indica tion 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 guidelin e 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;
M ean m easured concentrations : 0, 6.9, 10, 18, 27, and 48 μg a.i./L.Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

32 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 le vel and the controls. Test aquaria were labeled to identify the test sample nominal concentration and designated replicate. ” [Sprin g born
Life Science s , Inc. 1988d, p. 8

- 9]

“ The test was initiated when ten daphnids ( ≤ 24 hours old), twenty per treatment l evel and controls, were impartially selected and introduced to each exposure vessel. ” [Sprin g born Life
Science s , Inc. 1988d, p. 11]

Two control groups (dilution water control and solvent control) were tested concurrently. The number of immobilized daphni ds 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 t he 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 provid ed 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 concentratio ns 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. ” [Sprin g born Life
Science s , Inc. 1988d, p. 14]

The total hardness was 170

- 180 mg CaCO 3 /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

re moved 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 t he test was terminated.Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

33 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 d iluter. “ A dil uter stock solution of 1.10 mg A.I ./L was prepared by dilutin g 0.1102 g Anthraquinone (0.1100 g rams active ingredient [A.I.] ) with acetone to a total volume of 100 mL. The diluter was equipped with a 50

- mL gas

- tight Glanco R 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 h ighest 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).” [Sprin g born Life Science s , Inc. 1988d, p. 9

- 10]

Details on Sampling : “ Water samples were removed from both replicate ve ssels 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 vess els 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) fr om both replicate vessels of each test concentration and the controls. ” [Sprin g born Life
Science s , Inc. 1988d, p. 12

- 13]

Three quality assurance (QA) samples, formulated in test dilution water to contain
concentrations of the test substance unknown to t he 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 alka linity ranges as calcium carbonate (CaCO 3 ) 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 mi cromhos 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 calibra tion 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 EC 50 > 48 μg a.i./L (based on mean measured concentrations);
48

- hour NOEC = 48 μg a.i./L (based on mean measured concentrations).Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

34 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
immo bilization 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 th is [sic] data

, it was established that u nder 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 EC 5 0 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 No

Observed

- Effect Concentration established for this study was 48 μg A.I. /L, the highest mean
measured concentration tested

. ” [Springborn Life Sciences, Inc

. 1988d, p. 1 7 ]

STUDY RELIABILITY

[ 3 ] Not r eliable. 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 t he guid e line; (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 maxi mum 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 3 8 % of the measured maximum water solubi lity of 125 μg/L at a temperature and pH closely approximating the daphnid test conditions ( 2 2 °C and pH of 7. 2 )

.

REFERENCE S

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 Science s , Inc., 1988d.Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

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

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

Through 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 measureme nts 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

concentr ation; (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 )Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

36 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 aqua ria) 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 a nthraquinone 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 b y 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 a nthraquinon e 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 Anthraquin one.... Based on the results of the 0

- and 96

- hour analyses, the mean measured test concentrations of the three highest treatment level s 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/mi nute, which provided approximately six volume replacements every 24 hours. ” [Springborn Life Sciences, Inc

. 1988 e , 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 dilutedDraft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

37 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 ran ge. 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

concentrat ion present in the highest test concentration (86 μL acetone/L). ” [Springborn Life Sciences, Inc

. 1988 e , p.12

- 13]

Details on Sampling : “ The high, middle

, and low test concentrations (A and B replicates) were sampled and analyzed for Anth r aquinone (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 definiti ve 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. Al l 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 analyse s 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 samplin g 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 wate r 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 spong e s 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

. 1988 e , 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 u s ing 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 re quired 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 toDraft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

38 remove any new shell deposition. The exposure of oysters was initiated by impartially selecting and placing 20 oysters in each test aquarium (40 per treatm ent).” [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 produ ction 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 micromete r.” [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 EC 50 > 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 hour s 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 o f 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 t he 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) con trol 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 o f 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 EC 50 for this study was empirically estimated to be > 17 μg a.i./L, the highest concentration
of a nthraquinone tested

.Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

39 CONCLUSIONS

“ Based on th is [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 r eliable. 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 concentration s 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/h ou r/oyster instead of the flow rate of 1

- 5 L/h ou r/oyster recommend ed 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) m aximum 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 μ g /L was approximately 1 4 % of the measured maximum water solubility of
125 μg/L at a temperature and pH closely approximating the oyster test conditions ( 2 2 °C and pH of 7. 2 ) if the freshwater and saltwater solubility of the test substance are the same ; and (10) total organ ic carbon and particulate levels in the dilution water were not reported

.

REFERENCE S

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 Science s , Inc., 1988e.Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

40 C

- 3. Toxicity to Aquatic Plants

Summary of endpoint : Not a dequate . 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) a ccording 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 a lgae

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 compon ent of the growth medium, FeCl 3 , 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 no t specified and was not measured; (9) Whereas the duration of a study of Sel e nastrum 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 E1 218

- 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, s tates 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 o nly 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 initi al 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 requi rement 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, 2006 1 ) and 1.35 mg/L (25 °C; Yalkowsky and Dannenfelser, 1990 2 ).

There is uncertainty that the maintenance and growth medium used in this study included the
FeCl 3 that is present in the Synthetic Algal Nutrient Medium described by NERP/EPA (1971) and the freshwater algae m edium 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 th e 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 cove red with aluminum foil. Incubator temperature was 24 ± 1 °C with constant illumination from fluorescent
bulbs . Light intensity (lumens/m 2 ) was not specified. The initial pH value of the growth medium was not specified and pH was not measured over the cou rse 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 Aque ous Solubility Data. CRC Press, Boca Raton, FL (ISBN 0

- 8493

- 1532

- 8). Value obtained
from EPI Suite v4.1.Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

42 concentration of algae was 10 3 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 EC 50 (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 10 6 , 2.61 x 10 6 , and 2.46 x 10 6 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 10 6 , 8.13 x 10 6 , and 7.03 x 10 6 cells/mL at test concentrations of 0, 1, and 10 mg/L, r espectively. 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 EC 50 and NOEC values for inhibition of cell density of Sel e nastrum capricornutum were > 10 mg/L.

STUDY RELIABILITY

[3] Not r eliable

. 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, FeCl 3 , 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 Sel e nastrum 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 begi nning 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 exponenti al 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.Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

43 REFERENCE S

Ch illingworth, 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.
1 971. Algal Assay Procedure Bottle Test, August, 1971.Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

44 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 t his 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

- Anthra quinone

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 (Whatm an #41 filter paper) and diluted approximately. These compounds were then tested at 1, 10, and 100% saturation.” [Giddings 1979, p. 361 ]Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

45 1,4

- Anthraquinone “ was tested at three concentrations, with two replicate s 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 describ ed 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 10 7 cell s /mL, and 1 mL of this suspension was added to each test bottle. The bottles were place d on a rotary shaker at 100 rpm in an environmental chamber (24°C

... ). After 2 h, 0.2 mL of an NaH 14 CO 3 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 14 C 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 sample s was counted.” [Giddings 1979, p. 362]

RESULTS

Endpoi nt value(s): 4

- hour EC 50 > 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 guid eline : ( 1 ) the duration of this study was 4 hours

, whereas the duration of a study of Selenastrum capricornutum is typically 96 hours ; ( 2 ) p urity and composition of the test substance were not provided; ( 3 ) t est substance concentrations in growth media were not analytically monitored; ( 4 ) pH was not measured in growth media over the course of the study; ( 5 ) q uality parameters were not provided for the water used to prepare the growth medium; and ( 6 ) l ight intensity was not specified and was not measured

.Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

46 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

.Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

47 C

- 4. Acute Toxicity

Summary of endpoint : Adequate

Study 1 : Acute oral toxicity in r ats ( From ECHA website, Exp Key Acute toxicity: oral.001)

Title : Not provided

Th is 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 an d group No.2

– second step using the same dose. Animals wereDraft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

48 observed for 14 days following dosing. Body weight was measured before application, on the 8 th day of the study and at day 15, before euthanasia of animals. Observations for mortality and
clin ical examinations were performed daily. Gross necropsy of survivors was performed at study
termination.

Test animals

- Source: breeding farm VELAZ s.r.o., Kole č 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 day s

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) : LD 50 (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 LD 50 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 D irective 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 ca tegories 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 fin dings 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 examina tion in all animals.Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

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49 STUDY RELIABILITY

[2] Reliable with restrictions. The summary provide d limited information on the study methods
and results and did not include a full citation for the study report.

REFERENCE S

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 ackno wledged 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

50 Study 2: Acute inhalation toxicity in r ats ( 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 : 197 4

- 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/m 3 ( 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) : L C 50 > 244 mg/m 3 (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.Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

51 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 provide d limited information on the study methods
and results and did not include a full citation for the study report.

REFERENCE S

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).Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

52 C

- 5. Repeated

- Dose Toxicity

Summary of endpoint : Adequate

Study 1 : 14

- Week r epeated

- dose oral toxicity study in rats ( NTP, 200 5 )

Title : Toxicology and carcinogenesis studies of anthraquinone (CAS No. 84

- 65

- 1) in F344/N

rats and B6C3F1 mice (feed studies)

Th is 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 : No ne

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 equalityDraft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

53 and Tarone’s (1975) life table test to identify dose

- related tre nds. 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 a nimals with that site examined microscopically. For calculation of statistical significance, the incidences of most neopla sms

... 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 potenti al sites of occurrence (e.g., leukemia or lymphoma), the denominators
consist of the number of animals on which a necropsy was performed. ” S urvival

- adjusted neoplasm rate s for each group and each site

- specific neoplasm are also presented. The “ survival

ad justed 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 q uantal

- 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 t otal 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 h azard 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 anyw here 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

m ortality, was accommodated by adjusting the var iance 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. Continuity

corrected Poly

- 3 tests were u sed 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 normalDraft 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

- te st 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 Mann

Whitney U test ( Hollander and Wolfe, 1 973). Because vaginal cytology data are proportions (the proportion of the observation period that an animal was in a given estrous stage), an arcsine
transformation w as 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 fema le 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 aft er 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 30,00 0 ppm (equivalent to approximately 135, 275, 555, 1130, and 2350 mg/kg

bw /day) of anthraquinone for 14 weeks. “ Clinical pathology study groups of 10 male s and 10
female s 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 f rom 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 volum e, mean cell hemoglobin, and mean cell hemoglobin

c oncentration were determined using a Serono

- Baker System 9000 hematology analyzer (Serono

- Baker Diagnostics, Allentown, PA). Differential leukocyte counts and erythrocyte and platelet morphologies were de termined 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 ne w 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, so rbitol 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 cytolog y 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 wei ghed. 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 stud y 0 and 30,000 ppm rats ...” “ In addition to gross lesions and tissue masses, t he 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 examin ed in all lower exposure groups

. ”Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

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 s odium/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 m ean body weight s and body weight changes of all exposed female groups w ere less than in the controls. A bsolute and
relative l iver 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 a t ≥ 7500 ppm. Responsive a nemia 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, a n 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 a spartate 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

. B one 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 30,00 0 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 gla n d 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 TR

494. 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 r epeated

- dose oral toxicity study in mice ( NTP, 200 5 )

Title : Toxicology and carcinogenesis studies of anthraquinone (CAS No. 84

- 65

- 1) in F344/N rats and B6C3F1 mice (feed studies)

Th is 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 : No ne

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 5 300 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 val ues 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 s ite 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 “ survival

adjusted 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 fra ction 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

- Armitag e 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 giv en 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 vari ety 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. Continuity

corrected Poly

- 3 tests were used in the analysis of lesion incidence, and reported P val ues 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’sDraft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

60 multiple rang e 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) wa s 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 b y NTP personnel, and implausible values were eliminated from the analysis. Average severity values were analyzed for significance with the Mann

Whitney U test (Hollander and Wolfe, 1973). Because vaginal cytology data are proportions (the proportion of t he 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 varian ce (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 s elected 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 s tudy 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/kg

bw /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 sin us of all core study mice surviving to the end o f 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 er ythrocyte 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; hemoglobinDraft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

61 concentration; erythrocyte and platelet morphology; mean cell v olume; mean cell hemoglobin; mean cell hemoglobin concentration; and leukocyte count and differentials.

“ At the end of the 14

- week studies, samples were collected for spe rm motility eva luations 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 wer e 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 trim med, embedded in paraffin, sectioned to a thickness of 4 to 6 μm, and stai ned 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 glan dular), 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 increase d liver weight in both sexes

, responsive anemia in females

,
c ytoplasmic 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 l iver weight s w ere greater than in controls. A respo nsive 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 increas ed 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 incidence s and severity of centrilobular hypertrophy of the liver were significantly greater than in controls. Increased incidences of h ematopoietic 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). 200 5

. Toxicology and carcinogenesis studies of anthraquinone (CAS No. 84

- 65

- 1) in F344/N rats and B6C3F1 mice (feed studies). NTP TR

494. 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 r epeated

- dose oral toxicity study in rats ( NTP, 200 5 )

Title : Toxicology and carcinogenesis studies of anthraquinone (CAS No. 84

- 65

- 1) in F344/N rats and B6C3F1 mice (feed studies)

Th is 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 : 199 6

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 : No ne

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/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

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 tha t 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 ex amination 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 denomina tors 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 “ survival

adjusted rate (based on the Poly

- 3 method described below) account s 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 th e 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 assumption s. 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. Continuity

corrected 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’sDraft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

65 multiple range test for comparing all means to each other. Hemato logy, 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 t hat 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
f rom the analysis. Average severity values were analyzed for significance with the Mann

Whitney 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 estro us 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 s imultaneous 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 an d 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 mal e and five fe male 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 o f 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 ...”

“ Comple te necropsies and microscopic examinations were perfo rmed 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%Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

66 neutral buffered formalin, processed and trimmed, embedded in paraffin, sectioned to a thickness
of 5 to 6 μm, and stained with hematoxylin and eosin f or 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 : a drenal 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 glan d (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, thyro id 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 includ ed 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 t issues 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 diag noses 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 an alyses 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/kg

bw/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

. I ncidence s of mononu clear cell leukemia were less in exposed groups than in controls

.Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

67 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

exp osure, 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

- neoplas tic Effects at 2 Years : I n creased 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. Seve rities 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. T he 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 vacuola tion 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 a t 469, 938, and 1875 ppm and incidences of centrilobular hypertrophy were elevated at 938, 1875, and 3750 ppm.

I ncidences of congestion, pigmentation, and hematopoietic cell proliferation of the spleen were greater in all groups of exposed males and fem ales than in the controls. I ncidences 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 group s 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 blad der 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 incide ncesDraft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

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68 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 t here was s ome evidence of carcinogeni c activity of anthraquinone in male rats and clear evidence of carcinogenic activity in female rats. T he 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 with out 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 TR

49 4. September, 2005. Text in quotes is taken verbatim from NTP (2005).Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

69 Study 4 : 105

- Week r epeated

- dose oral toxicity study in mice ( NTP, 200 5 )

Title : Toxicology and carcinogenesis studies of anthraquinone (CAS No. 84

- 65

- 1) in F344/N
rats and B6C3F1 mice (feed studies)

Th is 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 : 199 6

Test species : B6C3F1 mice (male and female)

Number of animals/sex/dose : 50

Route of administration : Oral

– dietary

Vehicle : No ne

Exposure duration : 105 weeks

Doses/concentrations tested : 0, 8 33, 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 val ues for the survival analyses are two sided. ”Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

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70 “ 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 s ite 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 “ survival

adjusted 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 fra ction 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 neopl asm 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 i n 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 survi ved 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 le sions. 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 w ith controls and a test for an overall exposure

- related trend. Continuity

corrected 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 evaluatio ns, 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’sDraft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

71 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

- r elated 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 va lues were analyzed for significance with the Mann

Whitney 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 w as 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 a cross 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 toxicokineti c 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. Fiv e 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 t o 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 we eks, and at the end of the studies. ” “ Complete necropsies and microscopic
examinations were p erformed 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 step

sectioned at 1

- mm intervals, and additional sections were obtained from each kidney. ”Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

72 “ In addition to gross lesions and tissue mass es, 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, salivar y 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 st udies, 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 a nd 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 ge neral 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 d ose 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,
reviewi ng 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/kg

bw/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 co ntrol 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. I ncreased incidence s of fatty focal degeneration of he pato cytes were seen in males atDraft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

73 833 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. I ncreased
i ncidences of liver hematopoietic cell pro liferation and focal necrosis were seen only in males at 7500 ppm. I ncidences of intracytoplasmic inclusion body of the urinary bladder were increased in all exposed groups of males and females. I ncidences 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 750 0 ppm males than in
the c ontrol. 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 hepatocel lular neoplasms increased with a positive trend in male and female mice, and the incidences were increased in all exposed groups. The i ncidences 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 hi storical control

ranges... The i ncidences 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 t here was c lear 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 RELI ABILITY

[ 1 ] Reliable with out restrictions.

REFERENCE S

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 TR

494. September, 2005. Text in quotes is taken verbatim from NTP (2005).Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

74 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.

Th is 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), Friedel

Crafts technology (AQ

- FC) and Die ls

- 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 Butt erworth 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
laboratorie s 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 recrys talization 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 samp le 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 TestDraft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

75 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 tha t 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 s pecified 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 mutat ions 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 e nzyme 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 w as 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 WP 2 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 theDraft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

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76 appropriate vehicle control for test strains TA98, TA100 and WP2 uvrA and/or at least a 3

- fold
increase for tester strains TA1535 and T A1537.”

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

- chloroethyl amino propyl amino) acridine dihydrochloride (ICR

- 191) at 2.0 μg/plate in TA1537,
and 4

- nitroquinoline

- N

- oxide at 1.0 μg/plate in WP 2 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

- aminoanthracen e at 25 μg/plate in WP 2 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 n or 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. Mutagen ic 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 f or 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

- N A 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; Z eiger et al ., 1988) was unexpected because the data were inDraft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

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77 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
remain ing 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).Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

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78 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).

Th is 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 processes 1

. 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 oxid ation 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
D iels

- 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

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79 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 B utterworth 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. Eac h 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 m etabolic a ctivation : Sodium azide (TA100),
9

- aminoacridine (TA1537), 4

- nitro

- o

- phenylenediamine (TA98)

, and mytomycin

- C (TA102)
With m etabolic activation : 2

- ami noanthracene (all strains)Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

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80 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 f or 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 coloni es 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, TA10 0 , 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) show ed 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 concentratio ns 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.Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

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81 STUDY RELIABILITY

[1] Reliable without restrictions.

REFERENCE S

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 TR

494. 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.Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

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82 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.

Th is 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 accept ed 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 : M ouse 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 mutationDraft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

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83 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 criter ion 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 Contro l 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) : N ontoxic 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 tes t chemical did not induce forward gene mutations in L5178Y mouse lymphoma cells
either with or without metabolic activation.

STUDY RELIABILITY

[2] Reliable with restrictions. I mportant study details were missing including test guidelines followed, statistical methods used, negative control treatment, number of replicates, and volume
of test substance added.

REFERENCE S

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).Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

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84 C

- 7. Genetic Toxicity

– Chrom osomal Aberrations

Summary of endpoint : Adequate

Study 1 : In vitro c hromosomal 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.

Th is 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 a ccepted 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 sel ect 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 wi th 20.0 and 44.0 harvests in the confirmatory trials. No visual signs of cytotoxicity were observed in the cultures analyzed.Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

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85 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

- h our 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 con sidered 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. I mportant 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.

REFERENCE S

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).Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

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86 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

Th is 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 dos ed by oral gavage at up to 5000 mg/kg. Based on lack of significant toxicity, 5000Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

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87 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 (PCE s ). The ratio of polychromatic erythrocytes to normochromatic erythrocytes (PCE:NCE) was calculated to s ee 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 conditi ons 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 u sing 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 non

conservative trend test.Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

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88 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.

REFERENCE S

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).Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

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89 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).

Th is 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 test ed 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 di spersion 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 anthraqu inone dissolved in corn oil. The animals were killedDraft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

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90 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 score d for the frequency of micronucleated cells in each of five animals per dose group.”

Positive Control Substance : D imethylbenzanthracene 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 d ealt 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.

REFERENCE S

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 TR

494. September, 2005. Text in quotes is taken from NTP (2005).Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

91 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.Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

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92 Study 4 : In vivo m ouse 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).

Th is 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
abso lute 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 totalDraft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

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93 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 w as 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 frequenc ies 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 peripher al 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 gr oups, 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 increas ed 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.

REFERENCE S

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 TR

494. September, 2005. Text in quotes is taken from NTP (2005).Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

94 MacGregor, J.T., Wehr, C.M., Heni ka, 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. Fund am . Appl. Toxicol. 14:513

- 522.Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

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95 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 r ats

Th is 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 : R eproduction/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, a nd 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 forDraft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

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96 each specified endpoint and for all collection inte rv als. If Levene's test was not significant ( P > 0.01), a pooled estimate of the varia nce ( Mean Square Error or MSE) was computed from a one

way 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 Stud ent'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 pe rcent 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 weig ht 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.

T o 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 botto m 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 24 00 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 2Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

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97 weeks after ma ting 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

exam ination. 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 inc luded 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 counte d.

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 wer e 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 lac tation 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 n ot 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 d elivered, 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 calcula ted 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 u niform 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 r anged from 12.0 to 26.5% forDraft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

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98 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 w ere 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 a rt icle was detected in the control samples.

Test article

- related mortality was seen in females at the mid

- and high

- dose levels. In the mid

and high

- dose groups, 8/12 and 7/12 females, respectively, died or were euthanized in extremis ; most of these mo rtalities 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

- do se 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 l actation 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 cons umption 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 Week s 1

- 2 (premating period) for all groups of treated males, during Week s 5

- 7 (postmating period) for mid

- dose males, and during
Week s 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 f oci 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 notedDraft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

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- 1

99 in the liver and glandular stomach o f 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 fou r 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 copulato ry 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 lactat ion day 4. In the mid

- dose group, 10 females delivered, but only
two females retained li tt ers 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 lengtheni ng 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, resp ectively) 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 po st

- 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 livebo rn pups per litter in these groups was decreased with an
increase in stillbo rn pups. At the low dose, no effect of treatment was evident from parturition data, li tt er size, or pup survival to lactation day 4; the total number of pups born, as well as the

num bers 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 su rv ival 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 we ights 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 li tt ers rem ained on lactation day 4 for the mid

- dose group and, although statistical analysis could not be conducted due to the small li tt er size, weights were 51

- 58% lower than controls. Due to the small number of litters in the high

- dose group, statistical analysi s was not conducted for pup body weights for either time point, butDraft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

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100 weights on lactation day 0 were 2

- 7% lower than controls and weights on lactation day 4 were
10

- 1 1% lower than controls. The lower pup weights were considered indicative of a treatment

re lated 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
wei ghts 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 ani mal 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 stillbo rn 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 l actation 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 post

implantation loss per litter was observed at th e 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. Th e 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 livebor n 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).

ST UDY 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 b ody 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

sta tistically analyzed on a per

- pup or a per

- litter basis.Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

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101 REFERENCE S

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.Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

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102 D. SU PPLEMENTAL 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 a nd No. 9) were dosed in similar manner for aDraft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

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- 1

103 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 h ou r) : approximately 15 air changes per hour

- Photoperiod (h ou rs dark / h ou rs 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 ob ligatory 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 principl es. The summary did not provide a full citation for the study report.

REFERENCE S

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.Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

- 65

- 1

104 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

105 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 μ L 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 μ L 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. OD 570 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.Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

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106 Test Syst em : 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 EpiDerm TM 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 OD 570 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 : OD 570 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 viabilitiesDraft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

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107 CONCLUSIONS

Under the above

- described experimental design, the test substance was nonirritating for EpiDerm TM 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 resu lts. According to the MatTek instruction, a deviation occurred during this study when the 24

- hour preincubation period was omitted due to a time shortage.

REFERENCE S

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
dat e 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).Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

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- 1

108 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 fo llowing instillation.

Tool used to assess score : fluorescein and ophthalmoscopyDraft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

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109 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 h ou r) : approximately 15 air changes per hour

- Photoperiod (h ou rs dark/h ou rs 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 o f 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 al terations
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 a lterations
Cornea

– no alterationsDraft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

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110 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 v essels 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
val id GLP principles. The summary did not provide a full citation for the study report.

REFERENCE S

Study available from ECHA website, http://echa.europa.eu , first row of the “Search results” table resulting from a “Chem ical 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 Che micals 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

111 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 : I n 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 : W ater

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. OD 570 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 ET 50 value was calculated from slope of the line obtained.Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

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112 RESULTS

Endpoint value(s) : Non

- irritating

Results remarks : The mean OD 570 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 red uce MTT directly

Table 1 : OD 570 values obtained in the MTT test, their averages, standard deviations (%), and
relative viabilities
OD 570 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 min utes

, and 98.4% after 60 min utes of
administration. V iability did not decrease with time. The ET 50 value calculated was high

– 3.86*1020 min utes , which is higher than 60 min utes , showing that the test substance did not
af

fect the viability of the tissue and therefore

, did not have an irritation effect.

All criteria of assay acceptance have been met : OD 570 of negative control

- 1.719

– lies in interval 1.0 ≤ 1.759 ≥ 2.5, ET 50 for 0.3% Triton X

- 100 lies between 15 and 45 min utes (20.52 min utes ) and all OD 570 values fulfil l the condition average ± 15%.

CONCLUSIONS

In the experiment arrangement given above, the test substance was nonirritating in EpiOcularTM
model.

STUDY RELI ABILITY

[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 prin ciples, or present a clear interpretation of study
results.Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

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113 REFERENCE S

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 refer ence 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

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114 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 μ L 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 inDraft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

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115 ve hicular 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 K ladna, 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 h ou r) : approximately 15 air changes per hour

- Photoperiod (h ou rs dark / h ou rs light) : 12 hours light/dark cycle : 6am

- 6pm/6pm

- 6am

Range

- finding tests The high est 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 a ny 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 s ubstance 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.Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

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116 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

- paramet ric 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

reco rded 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 concentration

dependence. 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 expecte d mode of action as a contact allergen.

Based on these results, the test substance had a positive result in this LLNA test.Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

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117 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 posi tive 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.

REFERENCE S

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 r eference 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

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118 D

- 4. Genetic Toxicity

– Other

Study 1 : In vivo mouse DNA damage test (Cesaron e 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

- strand ed 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.Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

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119 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.

REFERENCE S

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.Draft HPV1 /HPV2 Chemical Data Adequacy Review ( May 2013 ) CASRN 84

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120 E. DATA ADEQ U ACY 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 No 1

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 Reportin g 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 t oxicity to aquatic plants endpoint at
this time.