SRC TR-10-048

HPV1 TEST RULE STUDY – DATA ADEQUACY ASSESSMENT

1-Chlorododecane, CAS No. 112-52-7

Combined Repeated-Dose/Reproductive/Developmental Toxicity

Prepared by:

Ashlee Aldridge, Justine Von Runnen and Joan D. Garey

Environmental Science Center
SRC, Inc.

7502 Round Pond Road North Syracuse, NY 13212

Contract No. EP-W-09-027

Task FG007.0.009.002

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

1-Chlorododecane, CAS No. 112-52-7

Combined Repeated-Dose/Reproduc tive/Developmental Toxicity

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 – Data Adequacy – Combined Repeated-Dose/Repro/Develop Tox CAS No. 112-52-7

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 – Data Adequacy – Combined Repeated-Dose/Repro/Develop Tox CAS No. 112-52-7

REPEATED DOSE/REPRODUCTIVE/ DEVELOPMENTAL TOXICITY

Title: A Combined 28-Day Repeated-Dose Oral Toxicity Study with the Reproduction/Developmental Toxicity Screen ing Test of 1-Chlorododecane in Rats

The study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: 1-Chlorododecane, CAS No. 112-52-7

Remarks: The test substance was a colorless to pale yellow liquid, stored at room temperature

and was stable for the duration of testing. Batch #N6227945, Purity 97.42%

METHOD

Method/guideline followed: The protocol was designed to be in general accordance with the US

EPA TSCA Guideline 40 CFR 799.9365 TSCA Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, 15 December 2000 and the OECD Guideline for Testing of Chemicals, Guidelin e 422, Combined Repeated Dose Toxicity Study with Reproduction/Developmental Toxi city Screening Test, 22 March 1996.

Test type: Combined repeated-dose with reproduc tive/developmental toxicity screening

GLP compliant?: Yes

Year study performed : 2008

Test species: Crl:CD(SD) rats from Charles River La boratories, Inc., Raleigh, NC. At the

initiation of dose administrati on (study day 0), the animals we re approximately 10 weeks old. Male body weights ranged from 326 g to 389 g. Female body weights on study day 0 ranged from 216 g to 273 g.

Number of animals/sex/dose: 10/sex/group

Route of administration: Oral gavage

Exposure duration: 32 to 52 Days

Doses/concentrations tested: 0 (vehicle control), 100, 300, 1000 mg/kg/day

Vehicle: Corn oil

Test method/conditions remarks: The test article, 1-Chlorododecane, in the vehicle, corn oil, was administered orally by gavage

2Draft HPV1 Test Rule – Data Adequacy – Combined Repeated-Dose/Repro/Develop Tox CAS No. 112-52-7

once daily to 3 groups of Crl:CD(SD) rats, each group consisting of 10 males and 10 females. Dosage levels were 100, 300 and 1000 mg/kg/day ad ministered at a dosage volume of 5 mL/kg. A concurrent control group of 10 rats/sex received the vehicle (corn oil) on a comparable regimen. Males received 14 daily doses prior to mating. Males were dosed throughout the mating period through 1 day prior to euth anasia for a total of 32 doses. Fe males received 14 daily doses prior to pairing and were dosed through lactation day 3 for a tota l of 39-51 doses; females with no evidence of mating/that failed to deliver were dos ed through the day prior to euthanasia (postmating or post cohabitation day 25) for a total of 39 or 52 doses. All an imals were observed twice daily for mortality and moribundity. Clinical observations, body weights and food consumption were recorded at appropriate intervals. Functional observational battery (FOB) and locomotor activity data were recorded for 5 males/group on study day 31 and for 5 females/group on lactation day 4. All F0 females were allowed to deliver and rear their pups until lactation day 4. F1 clinical observations and body weights were recorded on postnatal days (PND) 1 and 4. Pups were necropsied on PND 4. Clinical pathology evaluations (hematology and serum chemistry) were performed on 5 F0 animals/sex/group at necropsy. F0 males were euthanized following completion of the mating period (study day 32) and F0 females were euthanized on lactation day 4. Complete necropsies were conducted on all F0 animals, and selected organs were weighed. Se lected tissues were examined microscopically from all F0

animals.

All statistical tests were perfor med using appropriate computing devices or programs. Analyses were conducted using two-tailed tests (except as noted otherw ise) for minimum significance levels of 1% and 5%, comparing each test ar ticle-treated group to the control group by sex.

Data obtained from nongravid fema les were excluded from statistical analyses following the mating period. Where applicable, the litter was used as the experimental unit. Parental mating, fertility, conception and copulation indices were analyzed using the Chi-square test with Yates’ correction factor. Mean parental body weights (weekly, gestation and lactation), body weight changes and food consumption, offspring body we ights and body weight changes, gestation length, numbers of implantation sites and corpor a lutea, number of pups born, live litter size on PND 0, unaccounted-for sites, absolute and relativ e organ weights, clinic al pathology values (excluding differential white cell counts other than lymphocytes and neutrophils), pre-coital

intervals and FOB data values were subjected to a parametric one-way analysis of variance (ANOVA) to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett' s test was used to compare the test article-treated groups to the control group. FOB parameters that yield scalar or descriptive data in the test article-treated groups were compared to the control group using Fisher’s Exact test. Mean litter proportions (percent per litter) of males at birth and postnat al survival were subjected to the Kruskal-Wallis nonparametric ANOVA to determine intergroup differe nces. If the ANOVA revealed statistically significant (p<0.05) intergroup varian ce, Dunn’s test was used to co mpare the test article-treated groups to the control group. For locomotor activity, total and am bulatory counts were analyzed by sex and session, with a repeated measure an alysis of variance (RANOVA). Factors in the model included treatment group (TRT), time interval (TIME) and the interaction of time interval and treatment group (TRT*TIME). The SAS® pro cedure PROC MIXED was used for analysis

with the random effect of animal included as th e repeated measurement. The covariance structure across time was selected by comparing Ak aike’s Information Criterion (AIC).

3Draft HPV1 Test Rule – Data Adequacy – Combined Repeated-Dose/Repro/Develop Tox CAS No. 112-52-7

The monotonic dose-response relationship was evalua ted using sequential line ar trend tests based on ordinal spacing of dosage levels. The linear dose by time interaction (LinTrt*Time) was evaluated, and if significant at the 0.05 level, trend tests on treat ment means were performed at

the 0.05 level for each time interval . If the linear dose by time inter action was not significant, the trend test was conducted across the pooled ti me intervals by session only. Nonmonotonic dose responses were evaluated whenever no significant linear trends were detected but TRT and/or TRT*TIME interaction was signifi cant at the 0.01 level. Within the framework of the RANOVA, pair-wise comparisons were made for each in dividual treated group with the control group through linear contrasts. If TR T*TIME was significant, the comparisons were conducted for each time interval. If only the TRT effect wa s significant, the comparisons were conducted across the pooled time intervals of the en tire session. These nonmonotonic dose response comparisons were conducted at the 0.01 significance level.

All statistical analyses were conducted usi ng SAS version 8.2 (SAS Institute, Inc.,1999-2001)

software.

RESULTS

Endpoint value(s):

NOAEL F 0 (reproduction): 1000 mg/kg/day (highest dose tested)

LOAEL F 0 (reproduction): > 1000 mg/kg/day

NOAEL F 0 (systemic): 100 mg/kg/day

LOAEL F 0 (systemic): 300 mg/kg/day

NOAEL F 1 (developmental): 300 mg/kg/day

LOAEL F 1 (developmental): 1000 mg/kg/day

Results remarks: One female in the 1000 mg/kg/day group was euth anized in extremis on gestation day 21, an expected day of parturition. No evidence of partur ition was noted for this female; however, this female was hypoactive with eyes partially clos ed on the day of euthanasia. This morbidity around the time of parturition may be attributed to the stress of pregnancy exacerbated by the higher dose of test article resulting from the use of gestation day 20 body weight (dam plus fetal weights) for dose calculation. Another female in the 1000 mg/kg/day group had total litter loss on lactation day 1. This female had diminished nesting behavior on that day, which was probably due to an underlying pathologica l condition of the female (acute bacterial pyelonephritis). The diminished nesting behavior li kely contributed to poor conditi on of the pups, resulting in mortality/moribundity of the pups.

Salivation prior to dosing and cl ear material around the mouth (approximately 1 hour following dose administration) were noted in the 300 a nd/or 1000 mg/kg/day group males and females. These findings were considered to be related to test article treatment but are frequently

4Draft HPV1 Test Rule – Data Adequacy – Combined Repeated-Dose/Repro/Develop Tox CAS No. 112-52-7

observed in gavage studies and, ther efore, were not considered to be signs of toxicity. No test

article-related clin ical findings were noted in the 1 00 mg/kg/day group males and females. Lower mean body weight gains and food consumption were noted in the 1000 mg/kg/day group males during the first week of dose administra tion (study days 0-7). Lower mean body weight gains were also noted in th e 1000 mg/kg/day group males near the end of the study period, resulting in a mean body weight that was 6.0% lower than the cont rol group value at the end of the study. Mean body weight gain and food cons umption in the 1000 mg/kg/day group females were also slightly lower duri ng the first week of dose admini stration. However, the reductions were not of sufficient magnitude to result in lower mean body weights in these females. No adverse effects on mean body weights, body weight gains or food consumption were noted in the 100 and 300 mg/kg/day group males and females.

Male and female mating and fertility, male copulation and female conception indices and gestation length were not affected by test article administration at any dosage level. It could not be ascertained whether the female in the 1000 mg/ kg/day group that was euthanized in extremis on gestation day 21 was experiencing difficult labor because no signs of parturition were evident.

No test article-related effects were noted dur ing the FOB evaluations, and motor activity was unaffected by test article admini stration at all dosage levels.

There were no test article-re lated effects on hematology parameters in the 100, 300 and 1000 mg/kg/day group males and females. A higher mean alanine aminotransferase level was observed in the 1000 mg/kg/day group. Although a s light increase in hepatocellular hypertrophy was observed for this group, the increase in alanin e aminotransferase was of minimal magnitude and was not accompanied by increases in other hepa tic enzymes. Therefore, the relationship of

this increase to test article treatment was un certain and not considered adverse. Higher mean liver weights (absolute and relative to body a nd/or brain weight) occurred in both genders administered doses of 300 and 1000 mg/kg/day. These changes correlated with minimal to mild centrilobular hepatocellular hypertrophy, unaccompan ied by serum enzyme biomarkers of liver injury (except as noted above). Therefore, the changes in the liver we re considered to be consistent with, and likely a result of hepatic enzyme induction that was adaptive in nature, and

thus were not considered adverse. Pale liv ers noted macroscopically in the 1000 mg/kg/day group males corresponded to microvesicular vac uolation of periportal areas of the liver.

Additionally, higher mean adrenal gland weights (absolute, relati ve to final body weight and/or relative to brain weight) were observed for fema les in the 1000 mg/kg/day group relative to the control group. These increases correlated w ith the histologic findi ng of adrenocortical cytoplasmic vacuolation, considered to be a pot ential test article-relate d effect. Mean thymus gland weights were reduced in the 300 a nd 1000 mg/kg/day group females; there were no corresponding microscopic correlates. However, due to the magnitude of the change in these groups, (29% and 52%, respectively) and the dose -responsive nature of the decrease, the lower thymus gland weight in the 300 and 1000 mg/kg/da y group females was considered test articlerelated; however, the toxicologi cal significance of early physiologi cal involution of the thymus

in adult females is uncertain. There was no eviden ce of toxicity in the reproductive organs of

either sex.

5Draft HPV1 Test Rule – Data Adequacy – Combined Repeated-Dose/Repro/Develop Tox CAS No. 112-52-7

The mean numbers of corpora lutea, implantatio n sites and unaccounted-for sites in the test article-treated groups were sim ilar to those in the control gr oup. The mean numbers of pups born, live litter size on PND 0 and the percenta ge of males per litter in the 100, 300 and 1000 mg/kg/day groups were unaffected by test article administration.

Lower postnatal survival in the 1000 mg/kg/day group was obser ved during PND 0-1 and from birth to PND 4. Excluding the litter that was euthanized in extremis following diminished nesting behavior of the dam, these postnatal survival values remained lower than the minimum mean

value in the WIL historical control data. Lower mean F1 male and female birth weights (PND 1) and lower body weight gains were noted in th e 1000 mg/kg/day group comp ared to the control group values. There were no macroscopic findings in the F1 pups at the scheduled necropsy that

could be attributed to matern al test article administration.

CONCLUSIONS

Under the conditions of this screening study, 1000 mg/kg/day was considered to be the noobserved-adverse effect level (NOAEL) for reproductive toxicity of 1-chlorododecane when administered orally by gavage to Cr l:CD(SD) rats; however, the NOAEL for F 1 toxicity was 300 mg/kg/day based on lower postnatal survival and reduced pup body weights in the 1000 mg/kg/group. Lower mean body we ight gains and food consumpti on (primarily during the first week of dose administration) were noted at 1000 mg/kg/day, and morbidity in the 1 female and

total litter loss in another female were noted in this dose group near the time of parturition. In addition, lower mean thymus gland weights we re observed in the 300 and 1000 mg/kg/day group females. Therefore, the NOAEL for F 0 systemic toxicity was considered to be 100 mg/kg/day.

STUDY RELIABILITY

[1]-Reliable without restrictions

REFERENCE

EPA-HQ-OPPT-2005-0033-0314.4; Sloter , Eddie D. (2008) A Combin ed 28-Day Repeated Dose Oral Toxicity Study with the Reproductive/De velopmental Toxicity Screening Test of 1-

Chlorododecane in Rats. WIL Research Labor atories. Study Number WIL-636003. Sponsor:
Lonza, Inc.

6Review of TSCA Section 4 Final Study Report

CITB Project Manager: Catherine Roman

Chemical Name: 1-Chlorododecane

CAS Registry Number: 112-52-7

Report Title: A Combined 28-Day Repeated Dose Oral Toxicity Study with the Reproduction/Developmental Toxicity Screening Test of 1-Chlorododecane in Rats

Test Sponsor: Lonza, Inc.

Testing Lab: WIL Research Laboratories, LLC

Test Standard: U.S. EPA TSCA Guideline 799.9365

Date Study Received by EPA: September 19, 2008 Date Study Due to EPA: May 17, 2007

Special Instructions (e.g., regarding approved protocol modifications, etc.):

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

[D] 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.

[NA] 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.[D] 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. [X] 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 [ ] reasons in notes below).

Data Reliability 4. The study results are precise and accurate. [X] 5. Appropriate methods were used to determine [X] statistical significance. 6. If repeated, the test results would be expected to be the [X]

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):

Reviewer Name/Title/Bran ch/Division/Office: Ashlee Aldridge/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).