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 Work Assignment Manager 

March 5, 2010 
HPV1 Test Rule – Data Adequacy Assessment 

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

Combined Repeated-Dose/Reproductive/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 22202 
Draft HPV1 Test Rule – Data Adequacy – Combined Repeated-Dose/Repro/Develop Tox CAS No. 112-52-7

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 – 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 Screening 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, Guideline 422, Combined Repeated Dose Toxicity Study 

with Reproduction/Developmental Toxicity Screening Test, 22 March 1996. 

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

GLP compliant?: Yes 

Year study performed: 2008

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

initiation of dose administration (study day 0), the animals were 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

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Draft 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 administered 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 euthanasia for a total of 32 doses. Females received 14 daily doses 

prior to pairing and were dosed through lactation day 3 for a total of 39-51 doses; females with 

no evidence of mating/that failed to deliver were dosed through the day prior to euthanasia (postmating or post cohabitation day 25) for a total of 39 or 52 doses. All animals 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. Selected tissues were examined microscopically from all F0 

animals. 

All statistical tests were performed using appropriate computing devices or programs. Analyses 

were conducted using two-tailed tests (except as noted otherwise) for minimum significance 

levels of 1% and 5%, comparing each test article-treated group to the control group by sex. 

Data obtained from nongravid females 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 weights and body weight changes, gestation 

length, numbers of implantation sites and corpora lutea, number of pups born, live litter size 

on PND 0, unaccounted-for sites, absolute and relative organ weights, clinical 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 postnatal survival were subjected to the Kruskal-Wallis 

nonparametric ANOVA to determine intergroup differences. If the ANOVA revealed statistically 

significant (p<0.05) intergroup variance, Dunn’s test was used to compare the test article-treated 

groups to the control group. For locomotor activity, total and ambulatory counts were analyzed 

by sex and session, with a repeated measure analysis 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® procedure PROC MIXED was used for analysis 

with the random effect of animal included as the repeated measurement. The covariance structure 

across time was selected by comparing Akaike’s Information Criterion (AIC). 

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

The monotonic dose-response relationship was evaluated using sequential linear 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 treatment means were performed at 

the 0.05 level for each time interval. If the linear dose by time interaction was not significant, the 

trend test was conducted across the pooled time intervals by session only. Nonmonotonic dose 

responses were evaluated whenever no significant linear trends were detected but TRT and/or 

TRT*TIME interaction was significant at the 0.01 level. Within the framework of the RANOVA, 

pair-wise comparisons were made for each individual treated group with the control group 

through linear contrasts. If TRT*TIME was significant, the comparisons were conducted for 

each time interval. If only the TRT effect was significant, the comparisons were conducted 

across the pooled time intervals of the entire session. These nonmonotonic dose response 

comparisons were conducted at the 0.01 significance level. 

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

software. 

RESULTS

Endpoint value(s): 

NOAEL F0 (reproduction): 1000 mg/kg/day (highest dose tested) 

LOAEL F0 (reproduction): > 1000 mg/kg/day 

NOAEL F0 (systemic): 100 mg/kg/day 

LOAEL F0 (systemic): 300 mg/kg/day 

NOAEL F1 (developmental): 300 mg/kg/day 

LOAEL F1 (developmental): 1000 mg/kg/day 

Results remarks: 

One female in the 1000 mg/kg/day group was euthanized in extremis on gestation day 21, an 

expected day of parturition. No evidence of parturition was noted for this female; however, this 

female was hypoactive with eyes partially closed 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 pathological condition of the female (acute bacterial pyelonephritis). The 

diminished nesting behavior likely contributed to poor condition of the pups, resulting in 

mortality/moribundity of the pups. 

Salivation prior to dosing and clear material around the mouth (approximately 1 hour following 

dose administration) were noted in the 300 and/or 1000 mg/kg/day group males and females. 

These findings were considered to be related to test article treatment but are frequently 

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

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

article-related clinical findings were noted in the 100 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 administration (study days 0-7). Lower mean body weight 

gains were also noted in the 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 control group value at the end of 

the study. Mean body weight gain and food consumption in the 1000 mg/kg/day group females 

were also slightly lower during the first week of dose administration. 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 during the FOB evaluations, and motor activity was 

unaffected by test article administration at all dosage levels. 

There were no test article-related 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 slight increase in hepatocellular hypertrophy 

was observed for this group, the increase in alanine aminotransferase was of minimal magnitude 

and was not accompanied by increases in other hepatic enzymes. Therefore, the relationship of 

this increase to test article treatment was uncertain and not considered adverse. Higher mean 

liver weights (absolute and relative to body and/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, unaccompanied by serum enzyme biomarkers of liver 

injury (except as noted above). Therefore, the changes in the liver were 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 livers noted macroscopically in the 1000 mg/kg/day 

group males corresponded to microvesicular vacuolation of periportal areas of the liver. 

Additionally, higher mean adrenal gland weights (absolute, relative to final body weight and/or 

relative to brain weight) were observed for females in the 1000 mg/kg/day group relative to the 

control group. These increases correlated with the histologic finding of adrenocortical 

cytoplasmic vacuolation, considered to be a potential test article-related effect. Mean thymus 

gland weights were reduced in the 300 and 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/day group females was considered test articlerelated; however, the toxicological significance of early physiological involution of the thymus 

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

either sex. 

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

The mean numbers of corpora lutea, implantation sites and unaccounted-for sites in the test 

article-treated groups were similar to those in the control group. The mean numbers of pups 

born, live litter size on PND 0 and the percentage 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 observed 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 the 1000 mg/kg/day group compared to the control 

group values. There were no macroscopic findings in the F1 pups at the scheduled necropsy that 

could be attributed to maternal 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 Crl:CD(SD) rats; however, the NOAEL for F1 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 weight gains and food consumption (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 were observed in the 300 and 1000 mg/kg/day group 

females. Therefore, the NOAEL for F0 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 Combined 28-Day Repeated Dose 

Oral Toxicity Study with the Reproductive/Developmental Toxicity Screening Test of 1-

Chlorododecane in Rats. WIL Research Laboratories. Study Number WIL-636003. Sponsor: 

Lonza, Inc. 

6 
Review 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 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. [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 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): 

Reviewer Name/Title/Branch/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 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).