SRC TR-10-xxx 

HPV1 TEST RULE STUDY – DATA ADEQUACY ASSESSMENT 

9,10-Anthracenedione, CAS No. 84-65-1 

Reproductive/Developmental Toxicity Screening Test 

Prepared by: 

Teresa Manyin, Ashlee Aldridge, and Joan D. Garey 

Environmental Science Center 

SRC, Inc. 

7502 Round Pond Road 

North Syracuse, NY 13212 

Contract No. EP-W-09-027 

Task FG007.0.009.004 

Submitted to: 

OPPT/RAD/HPVCB (7403M) 

U.S. Environmental Protection Agency 

1200 Pennsylvania Avenue 

Washington, D.C. 20460 

Elizabeth Margosches, Project Officer 

Meena Sonawane, Work Assignment Manager 

David Brooks, Alternate Work Assignment Manager 

March 5, 2010 
HPV1 Test Rule – Data Adequacy Assessment 

9,10-Anthracenedione, CAS No. 84-65-1 

Reproductive/Developmental Toxicity Screening Test 

March 2010 

Submitted to: 

High Production Volume Chemicals Branch 

Risk Assessment Division 

Office of Pollution Prevention and Toxics 

Environmental Protection Agency 

WAM: Meena Sonawane 

Alternate WAM: David Brooks 

1200 Pennsylvania Avenue, NW 

Washington, DC 20460-0001 

Prepared By: 

SRC, Inc. 

Environmental Science Center 

2451 Crystal Drive, Suite 804 

Arlington, VA 22202 
Draft HPV1 Test Rule Study – Data Adequacy – Reproductive/Developmental Toxicity CAS No. 84-65-1

PREPARATION OF THE DATA ADEQUACY ASSESSMENT

This document contains the review of a toxicity study conducted in response to the first 

HPV TSCA Section 4 Test Rule (71 FR 13708, March 16, 2006). Using the applicable test 

guideline and professional judgment as guides, the study was reviewed for adequacy and 

evaluated for compliance with the test guideline. (The applicable test guideline is not always the 

test guideline required by the test rule, because some test sponsors requested and received 

approval from EPA to use a different guideline.) The study was summarized according to a 

standard format, assessed for adequacy, and assigned a Klimisch reliability code. If the study 

was determined to be inadequate, a basis for that conclusion is supplied. For studies already 

assigned Klimisch codes, an explanation is provided if the reviewer’s assessment of data 

adequacy differed from the pre-assigned Klimisch code. 

A detailed evaluation of the test report and study conduct is provided at the end of the 

summary document in a section entitled “Review of TSCA Section 4 Final Study Report”. Any 

deviations from the applicable test guideline are described. The study is also evaluated for 

compliance with Standard Operating Procedures, Good Laboratory Practice Standards, and 

Quality Assurance procedures, as well as the use of appropriate controls and test substances. 

Any deficiencies in the descriptions of the study method, test system, animals studied, physical 

conditions, dosage information, individual animal data, calculations, and statistical analyses are 

described, and internal inconsistencies within the study report are identified. An overall 

assessment is provided for each of the following: study conduct, data reliability, and data utility. 

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Draft HPV1 Test Rule Study – Data Adequacy – Reproductive/Developmental Toxicity CAS No. 84-65-1

REPRODUCTIVE/DEVELOPMENTAL TOXICITY 

Title: An Oral Reproduction/Developmental Toxicity Screening Study of Anthraquinone (CAS 

#84-65-1) in Rats 

The study is: Adequate Not Adequate 

Test substance formulations for the most part were neither homogenous nor were the intended 

concentration levels achieved; results are based on nominal concentrations rather than measured 

concentrations; numbers of corpora lutea, pre-, and post-implantation losses were neither 

measured nor reported. 

TEST SUBSTANCE 

 

Identity: 9,10-Anthracenedione 

Remarks: 99.4% purity; Batch 1543-I-GESU5099797, sampled from a commercial shipment; 

manufactured by the Friedel-Crafts process 

METHOD

Method/guideline followed: 40 CFR 799.9355 and OECD TG 421 

Test type: Reproductive/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 2 additional 

weeks for males or through lactation day 4 for females 

Doses/concentrations tested: 0, 150, 600 and 2400 mg/kg-bw/day (based on nominal 

concentrations) 

Vehicle: 1% guar gum in distilled water 

Statistical methods used: Treatment effects were analyzed using either analyses of variance 

(ANOVA) or Fisher’s exact test. For parameters analyzed by ANOVA, if sample sizes for all 

groups were 3 or greater, Levene's test was used to assess homogeneity of group variances for 

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Draft HPV1 Test Rule Study – Data Adequacy – Reproductive/Developmental Toxicity CAS No. 84-65-1

each specified endpoint and for all collection intervals. If Levene's test was not significant (p > 

0.01), a pooled estimate of the variance (Mean Square Error or MSE) was computed from a oneway ANOVA and pair-wise comparisons were made using a Dunnett's test to compare each 

treatment group with the control group. If there were only 2 groups involved, the Dunnett's test 

reduced to a Student's t-test. If Levene's test was significant (p < 0.01), comparisons with the 

control group were made using Welch's t-test with a Bonferroni correction. Groups with sample 

sizes less than 3 were excluded from statistical analysis. Data comprised of percent values were 

transformed using the arcsin of the square root before data analysis.

For parameters analyzed by Fisher’s exact test, an overall test for association between response 

and treatment was conducted using a Fisher's exact test. If this overall test was significant (p < 

0.05) and there were more than two groups, then a follow-up analysis was conducted where each 

treatment group was compared to the control group using a Fisher's exact test. 

To determine effects of treatment on pup body weight, an analysis of covariance was conducted 

on mean pup body weight per dam using litter size as a covariate. Pair-wise comparisons were 

conducted using Dunnett’s test. 

Test method/conditions remarks: 

Prior to initiation of test article administration, from each test batch, and from the third 

formulation preparation, duplicate samples (2.0 mL) of the dosing formulations at the 150 and 

2400 mg/kg-bw/day dose levels were collected to assess the homogeneity of the test article 

preparations employing the same method and batch size used during the study. The samples 

were collected from the top, middle, and bottom of the container, while stirring, using a syringe. 

Samples (2.0 mL) of the dosing formulations at each concentration were collected from Weeks 1, 

3, and 8 to assess the concentration of the test article. The samples were collected from the 

middle of the container, while stirring, using a syringe. 

Sprague-Dawley rats (12/sex/dose) were administered the test substance via gavage at 0, 150, 

600 or 2400 mg/kg-bw/day at a dose volume of 20 mL/kg. Control animals received the vehicle, 

1% guar gum in distilled water. Animals were exposed for 2 weeks prior to mating and during 

mating (up to 14 days); males were exposed for an additional 2 weeks after mating and females 

were exposed through gestation and through 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 to 90 

minutes post-dose), each animal was removed from the cage and given a detailed clinical 

examination. Necropsy examinations were performed on all adult animals euthanized in 

extremis or found dead, and all surviving animals at the end of the exposure period; special 

emphasis was placed on the organs of the reproductive system. Organs weighed included the 

epididymis, prostate gland, seminal vesicles, testes, ovaries, and uterus with cervix. Implantation 

sites in the uterus were counted. 

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Draft HPV1 Test Rule Study – Data Adequacy – Reproductive/Developmental Toxicity CAS No. 84-65-1

The litters were examined as soon as possible after delivery and parameters including litter size, 

the sex of the pups, number of dead and live born pups, and any gross abnormalities of the pups 

were recorded. Any intact pups found dead at birth were externally examined; stillborn status 

was determined by lung floatation in water. Any intact pups found dead during lactation were 

externally examined. Pups were weighed, sexed, and examined for external abnormalities on 

lactation day 0 and 4. On lactation day 4, the pups were euthanized via intraperitoneal injection 

of sodium pentobarbital solution, externally examined and any abnormalities were recorded. 

RESULTS

Endpoint value(s): The systemic LOAEL was 150 mg/kg-bw/day based on lower body weights 

in females and reduced food consumption in both males and females. The developmental 

LOAEL was 150 mg/kg-bw/day based on lower pup body weights on lactation day 4. NOAELs 

were not established for systemic and developmental toxicity. The reproductive LOAEL was 

600 mg/kg-bw/day based on decreases in the number of pups delivered, number of implantation 

scars, and pup body weight at birth, as well as an increase in length of the estrous cycle. The 

reproductive NOAEL was 150 mg/kg-bw/day. 

Results remarks: 

Preparation of the dosing suspensions was problematic. Due to the viscosity of the vehicle 

(1% Guar Gum), the formulations were unable to be stirred adequately, and therefore it was 

difficult to obtain uniform suspensions. Periodic concentration analysis of the test article 

suspensions prepared for study determined that the formulations for the most part were neither 

homogenous nor were the intended concentration levels achieved. Recoveries ranged from 66.0-

73.0% (Week 1), 59.5-84.7% (Week 3), and 25.1-99.5% (Week 8) in the treated groups. No test 

article was seen in the control samples. 

Test article-related mortality was seen in females at the 600 and 2400 mg/kg-bw/day levels. 

Eight and seven females in the 600 and 2400 mg/kg-bw/day groups, respectively, died or were 

euthanized in extremis; most of this mortality occurred during the gestation and lactation periods. 

One male in the 150 mg/kg-bw/day 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 at all treatment levels. Mean 

body weights of treated males were significantly lower than those of the controls throughout the 

mating and postmating periods at 600 and 2400 mg/kg-bw/day. Mean body weights of treated 

females at all dose levels were significantly lower than those of the controls throughout the 

premating, mating, gestation, and lactation periods. Gestation body weight gain was 

significantly lower over most intervals (gestation days 0-7, 7-14, 14-20, and 0-20) in the 600 and 

2400 mg/kg-bw/day groups. Body weight gain in the 150 mg/kg-bw/day group during gestation 

was not significantly different from controls. Body weight gain during lactation was not 

different from controls at any dose level. (Body weight changes for females during premating 

and mating periods were not reported.) 

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Draft HPV1 Test Rule Study – Data Adequacy – Reproductive/Developmental Toxicity CAS No. 84-65-1

A treatment-related effect on food consumption was evident in all treated males and females in 

comparison to controls, although these reductions were not dose-dependent. Food consumption 

for all treated males was significantly lower than controls during Week 1-2 (premating period) 

and was significantly reduced in males at the 600 mg/kg-bw/day level during Week 5-7 

(postmating period) and in the 150 mg/kg-bw/day males during Week 6-7. Food consumption 

during premating, gestation, and lactation was lower than controls in all treated female groups, 

except the 150 mg/kg-bw/day group during lactation. 

No test article-related organ weight changes were noted in rats of either sex. Test article-related 

macroscopic changes were noted in female rats, but not in males. Black or red foci were 

observed in the glandular stomach of several females at 600 and 2400 mg/kg-bw/day that died (3 

and 4 females, respectively). Also at 2400 mg/kg-bw/day, minimal focal necrosis and moderate 

periportal vacuolation of hepatocytes were observed in one female, edema of the stomach was 

seen in two of seven animals that died, and erosion/ulcer in the glandular stomach was seen in 

one female that died. Test article-related microscopic changes were noted in the liver and 

glandular stomach of female rats at 2400 mg/kg-bw/day, but not in males. 

All males and females in the treated groups were observed at one or more daily intervals with 

discolored urine (brown, orange, or red). In addition, decreased activity was observed in 4 and 5 

females in the 600 and 2400 mg/kg-bw/day groups, respectively, and 1 male in the 150 mg/kgbw/day group. 

The mating, fertility, and fecundity indices for treated males and females (83.3-100%) were 

similar to controls (91.7-100.0%). Likewise, the copulatory interval (days to mating) was 

unaffected by treatment and ranged from 2.4-3.1 days in the treated groups in comparison to 3.0 

days in the controls. All pregnant animals in the control and 150 mg/kg-bw/day groups delivered 

and retained litters through lactation day 4. In the 600 mg/kg-bw/day group, 10 females 

delivered, but only two females retained litters through lactation day 4. In the 2400 mg/kgbw/day group, four females delivered, but only one retained a litter through lactation day 4. 

Treatment did produce a lengthening of the estrous cycle; mean estrous cycle length (number of 

days per cycle) was increased in the treated females (4.7, 5.6, and 4.8 days in the 150, 600, and 

2400 mg/kg-bw/day groups, respectively) in comparison to controls (4.4 days), but the difference 

was only statistically significant at 600 mg/kg-bw/day. The total number of implantation scars 

per litter was significantly reduced at 600 and 2400 mg/kg-bw/day.

The number of pups delivered (live + dead) was significantly decreased in the 600 and 2400 

mg/kg-bw/day groups. The number of liveborn pups in these groups was decreased with an 

increase in stillborn pups. At 150 mg/kg-bw/day, no effect of treatment was evident from 

parturition data, litter size, or pup survival to lactation day 4; the total number of pups born, as 

well as the numbers of live and stillborn pups, were similar to controls and unaffected by 

treatment. 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 in the 150 mg/kg-bw/day 

group (89%) was comparable to the controls (97%). Pup survival to lactation day 4, although not 

dose-dependent, for the 600 and 2400 mg/kg-bw/day groups was significantly lower (16.5 and 

33 .3%, respectively) than that of the control. Very few pups survived to lactation day 4 in the 

2400 mg/kg-bw/day group, but no clinical observations were noted for these pups. Mean pup 

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Draft HPV1 Test Rule Study – Data Adequacy – Reproductive/Developmental Toxicity CAS No. 84-65-1

body weights (by sex and sexes combined) in the 150 mg/kg-bw/day group were similar to 

control at birth, but 8-10% lower by lactation day 4. In the 600 and 2400 mg/kg-bw/day groups, 

pup weights at birth and lactation day 4 were notably lower than control. In the 600 mg/kgbw/day group these weights were significantly lower (22-25%) in comparison to controls on 

lactation day 0. Only two litters remained on lactation day 4 for this group, and although 

statistical analysis could not be conducted due to the small litter size, weights were 51-58% 

lower than controls. Due to the small number of litters in the 2400 mg/kg-bw/day group, 

statistical analysis was not conducted for pup body weights, but weights on lactation day 4 were 

10-11% lower than controls. The lower pup weights at all dose levels were considered indicative 

of a treatment-related response. No macroscopic observations were evident in any pups 

(stillborn, died on study, or sacrificed on lactation day 4) at necropsy. 

CONCLUSIONS

Both male and female rats at all treatment levels were observed with discolored urine and 

decreased body weights and food consumption. Pup body weights on lactation day 4 were 

decreased at all treatment levels. Thus, the systemic and developmental LOAEL was 150 

mg/kg-bw/day. The reproductive LOAEL was 600 mg/kg-bw/day based on decreases in the 

number of pups delivered, number of implantation scars, and pup body weight at birth, as well as 

an increase in length of the estrous cycle. The reproductive NOAEL was 150 mg/kg-bw/day. 

STUDY RELIABILITY

[3] Not reliable. Test substance formulations for the most part were neither homogenous nor 

were the intended concentration levels achieved; results are based on nominal concentrations 

rather than measured concentrations; numbers of corpora lutea, pre-, and post-implantation 

losses were neither measured nor reported. 

REFERENCE

An Oral Reproduction/Developmental Toxicity Screening Study of Anthraquinone (CAS #84-

65-1) in Rats. MPI Research, Inc., Study No. 1271-001, February 6, 2007. 

6 
Review of TSCA Section 4 Final Study Report 

CITB Project Manager: Catherine Roman 

Chemical Name: 9,10-Anthracenedione 

CAS Registry Number: 84-65-1 

Report Title: An Oral Reproduction/Developmental Toxicity Screening Study of Anthraquinone (CAS #84-65-1) in 

Rats 

Test Sponsor: Chemical Products Corporation, Cartersville, GA 

Testing Lab: MPI Research, Inc., Mattawan, MI 

Test Standard: 40 CFR 799.9355 

Date Study Received by EPA: February 20, 2007 Date Study Due to EPA: May 17, 2007 

Special Instructions (e.g., regarding approved protocol modifications, etc.): None specified 

 

Part I. Test Report and Study Conduct

Enter one of the following codes into each of the checkboxes 1 through 13: Agree [A], Disagree [D], Not 

Applicable [NA], or Cannot Determine [C], and add comment, below, as needed

 [A] 1. The final study report indicates evidence of gross, unauthorized or undocumented deviation from 

 the EPA test standard requirements, including “shall,” “must,” or “will” violations. List 

 significant test standard deviations and “shall,” “must,” or “will” violations in comments below. 

 [D] 2. The final study report indicates evidence of deviations from the “should” statements in the study protocol 

that may have a bearing on the study conclusions. List these in comments below. 

 [D] 3. Approved modifications of test standard or testing protocol (CITB PM describe or attach) were not 

followed or are inadequately documented. 

 [D] 4. There is evidence that laboratory Standard Operating Procedures (SOPs) were inadequately followed. 

 [D] 5. The Statement of Quality Assurance (QA) inspections, their dates, and findings were missing or not signed. 

There is evidence that QA procedures may be inadequate. 

 [D] 6. There is evidence that Good Laboratory Practice Standards (GLPS) were inadequately followed. 

 [D] 7. The test, control, or reference substances are different from that specified in the section 4 test rule or 

Enforceable Consent Agreement (ECA). 

 [D] 8. Animal identification is inadequate or unclear; use of unhealthy animals or other factors (e.g., deviations 

from temperature, lighting, dosing) are apparent. 

 [D] 9. The final study report inadequately describes the study method. 

 [D] 10. The final study report inadequately describes the test system. 

 [A] 11. The final study report inadequately describes the dosage information. 
 [D] 12. The final study report does not accurately reflect the individual animal data (or that of other discrete 

observations pertinent to the type of study conducted), or individual data were not provided. 

 [D] 13. The final study report inadequately describes the calculations, interpolations, data transformations, 

statistical analyses, or modeling parameters used. 

Part II. Conclusions

Place a check mark beside the summary statement(s) that apply to this review and add comments below as 

appropriate

Part A. To be filled out by Reviewer

 

Study Conduct 1. The study was well-conducted. [ ] 

 2. The study was generally well-conducted, with some minor [ ] 

 deficiencies (list in notes below). 

 3. The study conduct was significantly deficient or questionable, (give [X] 

 reasons in notes below). 

Data Reliability 4. The study results are precise and accurate. [ ] 

 5. Appropriate methods were used to determine [X] 

 statistical significance. 

 6. If repeated, the test results would be expected to be the [ ] 

 same. 

Data Utility 7. The data are useful for risk assessment for the endpoint tested. [X] 

 8. The data are useful for some other purpose (e.g., dose-setting). [ ] 

 9. The data are not useful for any purpose (describe in notes below). [ ] 

Reviewer Comments (continue on separate page, if needed): 

“Must” violations: Numbers of corpora lutea, pre-, and post-implantation losses were neither measured nor reported. 

Dosage information: Test substance formulations for the most part were neither homogenous nor were the intended 

concentration levels achieved. Recoveries ranged from 66.0-73.0% (Week 1), 59.5-84.7% (Week 3), and 25.1-

99.5% (Week 8) in the treated groups. Additionally, the results are based on nominal concentrations rather than 

measured concentrations, and mean measured concentrations throughout the exposure period are not provided in the 

study report. 

Study conduct: The method of mixing the dosing solutions was questionable. 

Data reliability: Because the method of mixing dosing solutions was questionable, it is unclear whether the study 

results are precise and accurate and also if the test results would be the same if the study was repeated. 

Reviewer Name/Title/Branch/Division/Office: Teresa Manyin/SRC, Inc.

Reviewer Signature: David Brooks/HPVCB/RAD/OPPTS 

Part B. To be filled out by CITB PM

Tiered Testing Decision 1. The tiered testing trigger was met, additional testing is indicated. [ ] 

(when applicable) 2. The trigger for additional (tiered) testing was not met. [ ] 

 

Study Acceptability 3. The study is acceptable. [ ] 

 4. The study may not be acceptable [ ] 

 (provide rationale in memo to CITB chief).