SRC TR-10-xxx

HPV1 TEST RULE STUDY – DATA ADEQUACY ASSESSMENT

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

Reproductive/Developmental To xicity Screening Test

Prepared by:

Teresa Manyin, Ashlee Aldr idge, 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 Wo rk Assignment Manager

March 5, 2010HPV1 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 22202Draft HPV1 Test Rule Study – Data Adequacy – Reproductive/Developmental Toxicity CAS No. 84-65-1

PREPARATION OF THE DAT A ADEQUACY ASSESSMENT

This document contains the review of a toxi city 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 guide s, the study was reviewed for adequacy and evaluated for compliance with the test guideline. (T he applicable test guid eline 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 assi gned a Klimisch reliability code. If the study was determined to be inadequate, a basis for th at conclusion is supplie d. For studies already assigned Klimisch codes, an explanation is prov ided if the reviewer’s assessment of data adequacy differed from the pre-assigned Klimisch code.

A detailed evaluation of the test report a nd study conduct is provided at the end of the summary document in a section entitled “Revie w 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 Procedur es, Good Laboratory Prac tice Standards, and Quality Assurance procedures, as well as the use of appropriate controls and test substances. Any deficiencies in the descri ptions of the study method, test system, animals studied, physical conditions, dosage information, indi vidual 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.

1Draft 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 concen trations 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; ag e: ~ 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 us ing either analyses of variance (ANOVA) or Fisher’s exact test. For paramete rs analyzed by ANOVA, if sample sizes for all groups were 3 or greater, Levene's test was us ed to assess homogeneity of group variances for

2Draft HPV1 Test Rule Study – Data Adequacy – Reproductive/Developmental Toxicity CAS No. 84-65-1

each specified endpoint and for all co llection intervals. If Levene's test was not significant (p > 0.01), a pooled estimate of the variance (Mean Squa re Error or MSE) was computed from a oneway ANOVA and pair-wise comparisons were ma de using a Dunnett's test to compare each treatment group with the control group. If there were only 2 gr oups involved, the Dunnett's test

reduced to a Student's t-test. If Levene's te st 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 an alysis. 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 ove rall 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 contro l group using a Fisher's exact test.

To determine effects of treatment on pup body wei ght, 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 ar ticle administration, from each test batch, and from the third formulation preparation, duplicat e samples (2.0 mL) of the dosi ng 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 ba tch 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 concentra tion 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 lact ation 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, in jury, and the availabil ity of food and water twice daily throughout the duration of the study. Daily during tr eatment (approximately 30 to 90 minutes post-dose), each animal was removed fr om 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 repr oductive system. Organs weighed included the epididymis, prostate gland, seminal vesicles, testes , ovaries, and uterus with cervix. Implantation

sites in the uterus were counted.

3Draft HPV1 Test Rule Study – Data Adequacy – Reproductive/Developmental Toxicity CAS No. 84-65-1

The litters were examined as soon as possible afte r 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 birt h were externally exam ined; stillborn status was determined by lung floatation in water. A ny intact pups found dead during lactation were externally examined. Pups were weighed, sexe d, 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 soluti on, 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 bo th 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 devel opmental toxicity. The reproductive LOAEL was 600 mg/kg-bw/day based on decrease s in the number of pups deliv ered, number of implantation scars, and pup body weight at birt h, as well as an increase in le ngth of the estrous cycle. The reproductive NOAEL was 150 mg/kg-bw/day.

Results remarks: Preparation of the dosing suspen sions was problematic. Due to the viscosity of the vehicle (1% Guar Gum), the formulations were unable to be stirred adequatel y, and therefore it was difficult to obtain uniform suspensions. Periodi c concentration analysis of the test article suspensions prepared for study determined that th e formulations for the most part were neither homogenous nor were the intended concentratio n levels achieved. Rec overies ranged from 66.0- 73.0% (Week 1), 59.5-84.7% (Week 3), and 25.1-99.5% (W eek 8) in the treated groups. No test article was seen in the control samples.

Test article-related mortality was seen in fe males 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 gestati on and lactation periods. One male in the 150 mg/kg-bw/day group died, bu t in the absence of mortality among males at the higher dose levels and since macroscopic findings were unremarkable, this death was

considered unrelated to treat ment with anthraquinone.

Decreased body weights were observed in both male s 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 lo wer than those of the controls throughout the premating, mating, gestation, and lactation pe riods. Gestation body weight gain was significantly lower over most in tervals (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 dur ing lactation was not different from controls at any dose level. (Body weight changes for females during premating and mating periods were not reported.)

4Draft HPV1 Test Rule Study – Data Adequacy – Reproductive/Developmental Toxicity CAS No. 84-65-1

A treatment-related effect on food consumption wa s 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/d ay males during Week 6-7. Food consumption during premating, gestation, and lactation was lowe r 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 fe males at 600 and 2400 mg/kg-bw/day that died (3 and 4 females, respectively). Also at 2400 mg/k g-bw/day, minimal focal necrosis and moderate periportal vacuolation of hepato cytes were observed in one fema le, edema of the stomach was seen in two of seven animals that died, and eros ion/ulcer in the glandular stomach was seen in

one female that died. Test article-related mi croscopic 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 fo r treated males and females (83.3-100%) were similar to controls (91.7-100.0%). Likewise, th e 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 pre gnant 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 litt ers through lactation da y 4. In the 2400 mg/kgbw/day group, four females delivered, but only on e retained a litter thr ough 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, respecti vely) in comparison to controls (4.4 days), but the difference was only statistically significan t at 600 mg/kg-bw/day. The to tal number of implantation scars per litter was significantly reduc ed 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/d ay, no effect of trea tment 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 th e treated groups was considered comparable to controls and unaffected by treatment. Pup surviv al 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/d ay groups was significantly lower (16.5 and 33 .3%, respectively) than that of the control. Very few pups surv ived to lactation day 4 in the 2400 mg/kg-bw/day group, but no clinical observa tions were noted for these pups. Mean pup

5Draft HPV1 Test Rule Study – Data Adequacy – Reproductive/Developmental Toxicity CAS No. 84-65-1

body weights (by sex and sexes combined) in th e 150 mg/kg-bw/day group were similar to control at birth, but 8-10% lowe r by lactation day 4. In the 600 and 2400 mg/kg-bw/day groups, pup weights at birth and lactati on day 4 were notably lower than control. In the 600 mg/kgbw/day group these weights were significantl y 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 litt er size, weights were 51-58%

lower than controls. Due to the small numbe r 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 macrosc opic 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 le vels were observed with discolored urine and decreased body weights and food consumption. Pup body weights on lactation day 4 were

decreased at all treatment le vels. 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 implantati on 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.

6Review 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) inspect ions, their dates, and findings were missing or not signed. There is evidence that QA pr ocedures 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 summar y 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 ri sk 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 solu tions 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/Bran ch/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 me t, 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).