SRC TR-14-004

HPV3 TEST RULE DATA ADEQUACY REVIEW (FINAL):

REPEATED-DOSE, REPRODUCTIVE, AND DEVELOPMENTAL TOXICITY

Propanoic acid, 2-methyl-, 3-(benzoyloxy)-2,2,4-trimethylpentyl ester, CASRN 22527-63-5

Prepared by:

Megan Nelson, Amy Nguyen, Teresa Manyin, and William L. Richards

Defense and Environmental Solutions Division

SRC, Inc.

7502 Round Pond Road

North Syracuse, NY 13212

Contract No. EP-W-09-027

Task FG007.4.009.006

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

February 18, 2014
Final HPV3 Test Rule Data Adequacy Review (February 2014) CASRN 22527-63-5

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TABLE OF CONTENTS

A. SOURCE MATERIAL USED FOR ASSESSMENT .............................................................. 1

B. EVALUATING DATA ADEQUACY ..................................................................................... 1

C. SUMMARY OF DATA ADEQUACY FOR TESTING REQUIRED BY TEST RULE ........ 2

D. DATA FOR SIDS ENDPOINTS.............................................................................................. 4

D-1. Repeated-Dose, Reproductive, and Developmental Toxicity............................................ 4

E. DATA ADEQUACY SUMMARY TABLE........................................................................... 17
Final HPV3 Test Rule Data Adequacy Review (February 2014) CASRN 22527-63-5

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A. SOURCE MATERIAL USED FOR ASSESSMENT

This document evaluates a toxicity test for propanoic acid, 2-methyl-, 3-(benzoyloxy)-2,2,4-

trimethylpentyl ester (CASRN 22527-63-5) submitted by Eastman Chemical Company (dated 

December 17, 2013) to EPA in response to the final test rule, “Testing of Certain High 

Production Volume Chemicals; Third Group of Chemicals” (76 FR 65385, October 21, 2011; 

referred to herein as HPV3; available online at 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2009-0112-0086 as of 

February 18, 2014). 

The HPV3 test rule requires the following toxicological testing for CASRN 22527-63-5: C1 

(consisting of acute toxicity to fish, acute toxicity to Daphnia, and toxicity to algae testing if log 

Kow < 4.2 or chronic toxicity to Daphnia and toxicity to algae testing if log Kow ≥ 4.2), D (acute 

mammalian toxicity test), E1 (bacterial reverse mutation test), E2 (chromosomal aberration or 

micronucleus test), and F1 (combined repeated dose toxicity study with the 

reproduction/developmental toxicity screening test). Since the log Kow of CASRN 22527-63-5 

has been experimentally determined to be ≥ 5.4 (based on measured log Kow values of 5.4, 5.5, 

and 5.8 for the three main components of the test item)1, which is > 4.2, the C1 aquatic toxicity 

testing requirement is considered to include chronic toxicity to Daphnia and toxicity to algae 

testing. 

The December 17, 2013 submission2from Eastman Chemical Company (“Eastman”) contained 

the following toxicological test report:

 “Benzoflex 1046: Oral (Gavage) Combined Repeat Dose Toxicity Study with 

Reproduction/Developmental Toxicity Screening Test in the Rat (OECD 422 with 

Recovery Groups)”, submitted for the F1 testing requirement

These newly submitted data are evaluated for adequacy herein. No other toxicity data are 

evaluated in this report. 

B. EVALUATING DATA ADEQUACY

Data available for SIDS toxicity endpoints are evaluated for adequacy in Section D. The 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. If the data available 

for an 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 on the last page 

of this document (Section E).

 

1 Data for log Kow and other physicochemical properties of Benzoflex 1046 (a “bulk reaction product” containing 

CASRN 22527-63-5) were submitted to EPA on June 20, 2013 (available online at 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2009-0112-0145 as of February 18, 2014).

2 Submitted via email from L.W. Velsor (Eastman) to J. Schaeffer (EPA) on December 18, 2013.
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C. SUMMARY OF DATA ADEQUACY FOR TESTING REQUIRED BY TEST RULE

Justification for Use of Test Substance:

The test rule classifies propanoic acid, 2-methyl-, 3-(benzoyloxy)-2,2,4-trimethylpentyl ester 

(CASRN 22527-63-5) as a Class 1 Chemical Substance for which testing must be conducted 

with material having a purity of 99% or greater. However, in a letter to EPA dated February 20, 

2012 (available online at http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT2009-0112-0107 as of February 18, 2014), Eastman proposed the use of a bulk reaction product 

of the following composition: 60-65% propanoic acid, 2-methyl-, 3-(benzoyloxy)-2,2,4-

trimethylpentyl ester (CASRN 22527-63-5), 20-25% 1,3-pentanediol, 2,2,4-trimethyl-1,3-

dibenzoate (CASRN 68052-23-3), and 10-15% 2,2,4-trimethyl-1,3-pentanediol diisobutyrate 

(CASRN 6846-50-0). 

Eastman’s rationale for the use of this mixture (the “bulk reaction product”) was based on the 

fact that “The manufacture of benzoate ester for commercial use has been as a bulk reaction and 

there is no readily available process to synthesize the chemical substance by direct reaction to 

achieve the purity required for Class 1 substances. Eastman believes that use of the bulk reaction 

product which is formulated into finished products for commercial sale will more accurately 

represent exposure and use of the benzoate ester and avoid a costly and technically uncertain 

purification procedure.” Given that the major constituent (60-65%) of the bulk reaction product 

is the test rule substance, propanoic acid, 2-methyl-, 3-(benzoyloxy)-2,2,4-trimethylpentyl ester 

(CASRN 22527-63-5), and the remaining two major components (CASRNs 68052-23-3 and 

6846-50-0) can be considered close analogs of CASRN 22527-63-5, EPA approved Eastman’s 

plan to conduct aquatic and mammalian toxicity tests on this mixture via email from P. 

Campanella (EPA) to G. Shrum (Eastman) on April 19, 2012. This mixture, referred to as 

“Benzoflex 1046” in the submitted study reports, can be considered a supporting substance for 

CASRN 22527-63-5; as such, the toxicity of Benzoflex 1046 is expected to be representative of 

the toxicity of CASRN 22527-63-5.

Evaluation of Toxicological Data Submission Dated December 17, 2013:

Data submitted for the combined repeated dose toxicity study with the 

reproduction/developmental toxicity screening test for Benzoflex 1046, supplied by Eastman and 

reviewed herein, are adequate to satisfy the F1 testing requirement promulgated in the final 

HPV3 test rule. 

Evaluation of Previously Submitted Toxicological Data:

In a previous submission (dated February 26, 2013; available online at 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2009-0112-0138 as of 

February 18, 2014), Eastman supplied data from acute mammalian toxicity and gene mutation 

tests. These data were reviewed in a separate report (SRC Technical Report #13-050) and were 

determined to be adequate to satisfy the D and E1 testing requirements promulgated in the HPV3 

test rule.

In another previous submission (dated June 20, 2013; available online at 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2009-0112-0145 as of 
Final HPV3 Test Rule Data Adequacy Review (February 2014) CASRN 22527-63-5

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February 18, 2014), Eastman supplied data from chronic toxicity to Daphnia, toxicity to algae,

and chromosomal aberration tests. These data were reviewed in a separate report (SRC 

Technical Report #13-060). The data submitted for the chromosomal aberration test were 

determined to be adequate to satisfy the E2 testing requirement for CASRN 22527-63-5 

promulgated in the HPV3 test rule. However, the data submitted for the chronic toxicity to 

Daphnia and toxicity to algae tests were not considered adequate to satisfy the C1 testing 

requirement. 

Summary of Data Adequacy for Toxicological Endpoints:

Data submitted by Eastman for the chronic toxicity to Daphnia and toxicity to algae endpoints 

are not considered adequate; these tests need to be repeated. Adequate data have been submitted 

by Eastman for acute mammalian toxicity, repeated-dose toxicity, gene mutation, chromosomal 

aberrations, reproductive toxicity, and developmental toxicity endpoints. 
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D. DATA FOR SIDS ENDPOINTS

D-1. Repeated-Dose, Reproductive, and Developmental Toxicity

Summary of repeated-dose toxicity endpoint: Adequate

Summary of reproductive toxicity endpoint: Adequate

Summary of developmental toxicity endpoint: Adequate

Study 1: Combined repeated-dose oral toxicity study with the reproduction/developmental 

toxicity screening test in rats (Dunster, 2013)

Title: Benzoflex 1046: Oral (gavage) combined repeat dose toxicity study with 

reproduction/developmental toxicity screening test in the rat (OECD 422 with recovery groups)

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Benzoflex 1046; supporting substance for propanoic acid, 2-methyl-, 3-(benzoyloxy)-

2,2,4-trimethylpentyl ester (CASRN 22527-63-5)

Remarks: Clear, colorless liquid; Batch No. V046212201; Composition: 68.8% propanoic acid, 

2-methyl-, 3-(benzoyloxy)-2,2,4-trimethylpentyl ester (CASRN 22527-63-5), 19.5% 1,3-

pentanediol, 2,2,4-trimethyl-1,3-dibenzoate (CASRN 68052-23-3), 7% 2,2,4-trimethyl-1,3-

pentanediol diisobutyrate (CASRN 6846-50-0), 4.2% miscellaneous esters, 0.5% volatiles, and 

0.02% moisture.1

METHOD

Method/guideline followed: OECD TG 422 (1996) “Combined Repeated Dose Toxicity Study 

with the Reproduction/Developmental Toxicity Screening Test”

Test type: Combined Repeated-Dose Toxicity Study with the Reproduction/Developmental 

Toxicity Screening Test

GLP compliant?: Yes

Year study performed: 2013

 

1 The certificate of analysis provided in the study report describes the composition as follows: 68.8% “diester”, 

19.5% “dibenzoate”, 7% “TXIB”, 4.2% miscellaneous esters, 0.5% volatiles, and 0.02% moisture. In an email dated 

July 3, 2013 from G. Shrum (Eastman) to J. Schaeffer (EPA), Eastman indicated that “diester” was synonymous 

with propanoic acid, 2-methyl-, 3-(benzoyloxy)-2,2,4-trimethylpentyl ester (CASRN 22527-63-5), “dibenzoate” was 

synonymous with 1,3-pentanediol, 2,2,4-trimethyl-1,3-dibenzoate (CASRN 68052-23-3), and “TXIB” was 

synonymous with 2,2,4-trimethyl-1,3-pentanediol diisobutyrate (CASRN 6846-50-0).
Final HPV3 Test Rule Data Adequacy Review (February 2014) CASRN 22527-63-5

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Test species: Wistar HanTM:RccHanTM:WIST strain rats; obtained from Harlan Laboratories 

UK, Ltd., Blackthorn, Bicester, Oxon, UK; rats were approximately 12 weeks old and weighed 

295–346 grams (males) and 182–212 grams (females) at the start of testing. 

Number of animals/sex/dose: 12 (non-recovery animals); 5 (recovery animals)

Route of administration: Oral – gavage

Vehicle: Arachis oil BP

Exposure duration: 

Non-recovery males: 42 days; Non-recovery females: 41–46 days (throughout 14 days of premating, mating [maximum of 14 days], gestation, and up to postpartum/lactation day 4); 

Recovery animals: 42 days (males and females), followed by 14 days without treatment

Doses/concentrations tested: Non-recovery groups: 0, 30, 60, and 120 mg Benzoflex 1046/kgbw/day; Recovery groups: 0 and 120 mg Benzoflex 1046/kg-bw/day

Statistical methods used: “Where considered appropriate, quantitative data... [were] subjected 

to statistical analysis to detect the significance of intergroup differences from control; statistical 

significance was achieved at a level of p < 0.05. Statistical analysis was performed on the 

following parameters: 

Grip Strength, Motor Activity, Body Weight, Body Weight Change, Food Consumption during 

gestation and lactation, Water Consumption during gestation and lactation, Pre-Coital Interval, 

Gestation Length, Litter Size, Litter Weight, Sex Ratio, Corpora Lutea, Implantation Sites,

Implantation Losses, Viability Indices, Offspring Body Weight, Offspring Body Weight Change,

Offspring Surface Righting, Hematology, Blood Chemistry, Absolute Organ Weights, Body 

Weight-Relative Organ Weights.” [Dunster 2013, p. 37]

Calculations of group means and comparisons among groups sometimes excluded values 

obtained from females that did not become pregnant, those that became pregnant but did not 

deliver, and/or those that exhibited total litter loss.

“Data were analyzed using the decision tree from the ProvantisTM Tables and Statistics Module 

as detailed as follows: 

Where appropriate, data transformations were performed using the most suitable method. The 

homogeneity of variance from mean values was analyzed using Bartlett’s test. Intergroup 

variance... [was] assessed using suitable ANOVA [analysis of variance], or if required, 

ANCOVA [analysis of covariance] with appropriate covariates. Any transformed data were 

analyzed to find the lowest treatment level that showed a significant effect using Williams’ test 

for parametric data or the Shirley test for non-parametric data. If no dose response was found 

but the data show non-homogeneity of means, the data were analyzed by a stepwise Dunnett’s 

(parametric) or Steel (non-parametric) test to determine significant difference[s] from the control 

group. Where the data were unsuitable for these analyses, pair-wise tests... [were] performed 
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using the Student[’s] t-test (parametric) or the Mann-Whitney U test (non-parametric).” [Dunster 

2013, p. 37]

“Data not analyzed by the Provantis data capture system were assessed separately using the 

SPSS statistical package. Initially, the homogeneity of the data was assessed using Levene’s 

test. Where Levene’s test was shown to be non-significant (p ≥ 0.05), parametric analysis of the 

data was applied, incorporating analysis of variance (ANOVA). If... [these] data... [were]

shown to be significant, this analysis was followed by pair-wise comparisons using Dunnett’s 

test. Where Levene’s test was significant, non-parametric analysis of the data was analyzed 

incorporating the Kruskal-Wallis test which if significant, was followed by the Mann-Whitney U 

test. Dose response relationship[s]... [were] also... investigated by linear regression. Where the 

data... [were] unsuitable for these analyses, then pair-wise tests were performed using the 

Student[’s] t-test (parametric) or the Mann-Whitney U test (non-parametric).” [Dunster 2013, p. 

37]

“Due to the preponderance of non-normally distributed data, reproductive parameters 

(implantations losses, offspring sex ratio, and offspring surface righting) were analyzed using 

non-parametric analyses.” [Dunster 2013, p. 37]

The standard unit of assessment for analyses of pup data was the litter.

Test method/conditions remarks: 

Test Item Preparation: 

The test item was diluted in Arachis oil BP to prepare test concentrations. Stability and 

homogeneity analyses of test item formulations in Arachis oil BP stored at 4 °C in the dark 

showed the test item to be stable for at least 16 days under these conditions. Therefore,

formulations were prepared twice per month during the toxicity study and stored at 

approximately 4 °C in the dark.

Analyses of Test Item Formulations: 

Test item concentrations in dosing formulations (nominal concentrations of 0, 7.5, 15, and 30 

mg/mL, corresponding to doses of 0, 30, 60, and 120 mg Benzoflex 1046/kg-bw/day, 

respectively) were analyzed in samples collected weekly. Test item concentrations in the dosing 

formulations were determined by high performance liquid chromatography (HPLC) with UV 

detection (wavelength of 231 nm).

Experimental Procedures:

Following a 6-day acclimatization period, groups of rats (12/sex/group) were orally administered 

the test item daily via gavage at dose levels of 30, 60, or 120 mg Benzoflex 1046/kg-bw/day in 

Arachis oil BP. A control group was treated with the vehicle only. All animals were dosed at a 

consistent dosing volume of 4 mL/kg-bw. Treatment continued throughout the pre-mating (14 

days), mating [maximum of 14 days], and post-mating periods, until scheduled sacrifice. The 

animals were treated for a total of 42 consecutive days for males or through postpartum day 4 for 

females (41–46 consecutive days), with the exception that females were not treated during 

parturition. 
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Prior to mating, animals were housed as groups or 4 or 5/sex. During the mating period, animals 

were paired (1 male: 1 female) for up to 14 days until there was evidence of mating (the presence 

of a copulation plug or the presence of sperm in a vaginal smear). Following evidence of mating

(designated as day 0 post coitum), males were returned to their original cages. Mated females 

were housed individually (or with their surviving pups after parturition) throughout gestation and 

lactation. Food and water were available ad libitum throughout the study.

Males were euthanized on day 43. Females were allowed to deliver and rear their pups until 

postpartum (lactation) day 5, when all females and their pups were euthanized (day 42 to 47). 

Non-pregnant females were euthanized on day 25 post coitum. 

Recovery groups:

Additional groups of rats (5/sex/group) were administered the test item via gavage at 0 or 120 

mg Benzoflex 1046/kg-bw/day for 42 days concurrently with the non-recovery groups. 

Recovery animals were observed for two weeks beyond the termination of treatment. Recovery 

animals were housed as groups of 5/sex throughout the treatment period and were not mated. All 

recovery animals were euthanized at the end of the two-week recovery period (day 57).

Clinical Observations: 

All non-recovery and recovery animals were examined for overt signs of toxicity, ill-health, and 

behavioral changes immediately prior to dosing, and two to three times after each daily dose. 

Recovery animals were observed daily during the 2-week recovery period. 

Body Weight and Food/Water Consumption:

Individual body weights of non-recovery animals were recorded on day 1 (prior to dosing), at 

least weekly throughout the treatment period, and at the time of euthanasia. Individual body 

weights of mated non-recovery females were recorded on post coitum days 0, 7, 14, and 20, and 

on postpartum days 1 and 4. Individual body weights of all recovery animals were recorded on 

day 1 (prior to dosing), weekly throughout the treatment and recovery periods, and at the time of 

euthanasia. 

“During the pre-pairing period, weekly food consumption was recorded for each cage of [nonrecovery] adults. This was continued for males after the mating phase. For females showing 

evidence of mating, food consumption was recorded for the periods covering post coitum days 

0–7, 7–14, and 14–20. For females with live litters, food consumption was recorded on days 1 

and 4 post partum. Weekly food... [consumption was determined] for each cage of recovery 

group animals throughout the study period.” [Dunster 2013, p. 29] 

Water intakes of all animals were measured daily throughout the study, except during the pairing 

phase of non-recovery animals.

“Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively 

for non-recovery males (except during the mating phase) and recovery group animals throughout 

the study period and for non-recovery females during the pre-pairing phase. Due to offspring 

growth and milk production, food efficiency could not be accurately calculated for females 

during gestation and lactation.” [Dunster 2013, p. 29]
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Neurological Assessments:

All non-recovery animals were observed for signs of behavioral toxicity on the day prior to the 

first day of treatment and at weekly intervals throughout the study. Behavioral assessments 

evaluated gait, tremors, twitches, convulsions, bizarre/abnormal/stereotypic behavior, salivation, 

piloerection, exophthalmia, lachrymation, hyper/hypothermia, skin color, respiration, palpebral 

closure, urination, defecation, transfer arousal, and tail elevation. 

Functional performance tests and sensory reactivity tests were performed on five adult 

animals/sex from each non-recovery group on day 38 for males or postpartum day 4 for females. 

Functional performance tests evaluated motor activity and forelimb/hindlimb grip strength. 

Sensory reactivity tests examined reactivity to auditory, visual, and proprioceptive stimuli, 

including grasp response, vocalization, touch escape, pupil reflex, toe pinch, tail pinch, finger 

approach, blink reflex, and startle reflex.

Hematology and Clinical Chemistry Analyses:

Blood samples for hematology and clinical chemistry evaluations were collected from 5 adult 

animals/sex from each non-recovery group, and from all recovery animals, on the day prior to 

euthanasia. Hematological parameters that were evaluated included hemoglobin, erythrocyte 

count (i.e., red blood cell count), hematocrit, erythrocyte indices (mean corpuscular hemoglobin, 

mean corpuscular volume, and mean corpuscular hemoglobin concentration), total leukocyte 

count (i.e., white blood cell count), differential leukocyte count (including counts for neutrophils, 

lymphocytes, monocytes, eosinophils, and basophils), platelet count, prothrombin time, and 

activated partial thromboplastin time. The following clinical chemistry parameters were 

determined from plasma collected from each blood sample: urea, glucose, total protein, albumin, 

albumin/globulin ratio, sodium, potassium, chloride, calcium, inorganic phosphorus, aspartate 

aminotransferase, alanine aminotransferase, alkaline phosphatase, creatinine, total cholesterol, 

total bilirubin, and bile acids. 

Urinalysis:

Urinalysis was performed on 5 males/group from each non-recovery group during the last week 

of treatment. Urinalysis was performed on all recovery males during the final week of recovery. 

Urinalysis parameters included volume, specific gravity, pH, protein, glucose, ketones, bilirubin, 

urobilinogen, blood, and appearance.

Gross Necropsy and Histopathology:

All adults, including those that died during the study, were subjected to a full external and 

internal examination and selected organs were weighed. Absolute organ weights were recorded 

for 5 adult animals/sex/group from each non-recovery group and for all recovery group animals 

for the following organs: adrenals, brain, heart, kidneys, liver, spleen, thymus, and 

thyroid/parathyroid (post-fixation). Absolute organ weights were recorded for all non-recovery 

and recovery animals for the following organs: epididymides, ovaries, pituitary gland (postfixation), prostate gland, seminal vesicles, testes, and uterus with cervix. Relative organ weights 

were calculated for each animal as the ratio of absolute organ weight to individual body weight. 
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The following tissues collected from 5 non-recovery rats/sex from the control and high-dose 

groups were examined microscopically: adrenals, aorta (thoracic), bone and bone marrow (femur 

including stifle joint), bone and bone marrow (sternum), brain (including the cerebrum, 

cerebellum, and pons), cecum, colon, duodenum, eyes, heart, ileum (including Peyer’s patches), 

jejunum, kidneys, liver, lungs (with bronchi), lymph nodes (mandibular and mesenteric), muscle 

(skeletal), pancreas, esophagus, rectum, salivary glands, sciatic nerve, skin (hind limb), spinal 

cord (cervical, mid-thoracic, and lumbar), spleen, stomach, thyroid/parathyroid, trachea, thymus, 

and urinary bladder. 

The following tissues collected from all non-recovery animals from the control and high-dose 

groups, any infertile males, and all females that did not give birth were examined 

microscopically: gross lesions, coagulating gland, epididymides, mammary gland, ovaries, 

pituitary gland, prostate gland, seminal vesicles, testes, uterus/cervix, and vagina. 

Reproductive and Developmental Toxicity:

Pregnant females were observed daily and the following parameters were recorded: date of 

pairing, date of mating, date and time of observed start of parturition, and date and time of 

observed completion of parturition. Upon the completion of parturition (designated as 

postpartum day 0), the numbers of live and dead offspring, and the sex of the offspring, were

recorded. All live offspring were evaluated for surface righting reflex on postpartum day 1. 

The following parameters were recorded for each litter during the postpartum period: number of 

offspring alive (recorded daily; reported on postpartum days 1 and 4), sex of the offspring on 

postpartum days 1 and 4, clinical condition of the offspring daily from birth to postpartum day 5, 

and individual offspring weights on postpartum days 1 and 4. Calculated mating, fertility, 

parturition, and offspring parameters included pre-coital interval, mating index, pregnancy index, 

gestation length, parturition index, live birth index, viability index, sex ratio, mean litter weight

(calculated retrospectively from individual offspring weights), mean offspring weight, and mean 

offspring body weight change. 

At necropsy, reproductive organs of adult animals were weighed and histological examinations 

were conducted on reproductive tissues, as described above. 

The uterus of each non-recovery female was examined for signs of implantation. The numbers

of implantations in each uterine horn and corpora lutea were recorded and used to calculate the 

pre- and post-implantation loss. “Detailed qualitative examination of the testes was undertaken, 

taking into account the tubular stages of the spermatogenic cycle. The examination was

conducted in order to identify treatment-related effects such as missing germ cell layers or types, 

retained spermatids, multinucleated or apoptotic germ cells, and sloughing of spermatogenic 

cells into the lumen. Any cell- or stage-specificity of testicular findings... [were] noted.” 

[Dunster 2013, p. 34]

Endpoint value(s):

Systemic Toxicity:

LOAEL (female, systemic toxicity) = 30 mg/kg-bw/day, based on reduced mean body weight 

during gestation and lactation, reduced mean body weight gain during the pre-pairing period, and 
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reduced mean food consumption during gestation; NOAEL (female, systemic toxicity) = Not 

established

LOAEL (male, systemic toxicity) = 120 mg/kg-bw/day, based on effects on clinical chemistry 

(reduced mean urea and reduced mean albumin/globulin ratio) and increased mean absolute and 

relative kidney and liver weights; NOAEL (male, systemic toxicity) = 60 mg/kg-bw/day

Reproductive Toxicity:

LOAEL (reproductive toxicity) = 120 mg/kg-bw/day, based on an increase in total litter loss and 

a reduced number of litters reared until lactation day 5; NOAEL (male and female, reproductive 

toxicity) = 60 mg/kg-bw/day

Developmental Toxicity: 

LOAEL (developmental toxicity) = 120 mg/kg-bw/day, based on an increase in total litter loss

and a reduced number of litters reared until lactation day 5; NOAEL (developmental toxicity) = 

60 mg/kg-bw/day

Results remarks:

Analyses of Test Item Formulations:

Measurement of test item concentrations by HPLC in formulations prepared for dosing showed 

that formulations contained 98–104% of nominal concentrations.

Mortality and Clinical Observations:

No mortalities occurred and no treatment-related clinical signs were observed in adult nonrecovery or recovery animals. “Clinical signs were confined to the presence of post-dose 

increased salivation... [in all] animals of either sex treated with 120 mg/kg-bw/day. Isolated 

instances of increased salivation following dosing were also evident at 60 mg/kg-bw/day.

Increased salivation is often recorded following the oral administration of an unpleasant tasting 

and/or locally irritant test item formulation and, in isolation, is considered not to be indicative of 

systemic toxicity.” [Dunster 2013, p. 39]

Body Weight:

Non-recovery groups:

At all doses, female group mean body weights were statistically significantly (p < 0.01) reduced 

at all time points throughout gestation (measured on gestation days 0, 7, 14, and 20), with 

reductions of 8.0 to 17.4% relative to the control group. Female body weights at all doses were

also statistically significantly (p < 0.01) reduced at both time points during lactation (measured 

on lactation days 1 and 4), with reductions of 9.2 to 12.5% relative to the control group. The 

decreases in female body weight during gestation and lactation exhibited a dose-related trend.

No significant differences were observed in the body weights of females during the pre-mating 

period or in the body weights of males throughout the study.

Female group mean body weight gains were statistically significantly (p < 0.01) reduced during 

pre-pairing days 1–8 at 60 and 120 mg/kg-bw/day (reductions of 127.7% and 121.5%, 

respectively, at these doses relative to the control group). Female body weight gains were 
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statistically significantly (p < 0.01) reduced during pre-pairing days 8–15 at 30, 60, and 120 

mg/kg-bw/day (reductions of 61.0%, 40.7%, and 74.8%, respectively, at these doses relative to 

the control group). 

Female cumulative body weight change during gestation days 0–20 was statistically significantly 

reduced at 60 (p < 0.01) and 120 (p < 0.001) mg/kg-bw/day, with reductions of 24.2% and 

34.2%, respectively, relative to the control group.

No significant differences in body weight gain were observed in females during lactation or in 

non-recovery males during any interval of the exposure period.

Recovery groups:

No statistically significant differences in body weight were observed in males or females of 

recovery groups. Group mean body weight gain was statistically significantly (p < 0.05) reduced 

in recovery group males at 120 mg/kg-bw/day during the first, second, and final week of the 

study, compared to control males (reductions of 90.8%, 37.3%, and 137.5% were observed 

during days 1–8, 8–15, and 50–57, respectively). Since no statistically significant reductions 

were observed in the body weight of males in recovery and non-recovery groups and no 

statistically significant reductions in body weight gain were seen in non-recovery males, the 

toxicological relevance of the body weight gain reductions in recovery group males at 120 

mg/kg-bw/day is not clear.

Recovery group females treated with 120 mg/kg-bw/day lost weight (group mean loss of 2.4 g 

versus a group mean gain of 4.8 g for the control group) during the first interval of the study

(days 1–8); the reduction in group mean body weight gain during this time period (150% relative 

to the control group) was statistically significant (p < 0.05). Group mean body weight gain in 

recovery females treated with 120 mg/kg-bw/day was comparable to the control group for the 

remainder of the treatment period and the recovery period.

Food/Water Consumption and Food Efficiency:

Group mean food consumption did not appear to be affected by treatment in exposed nonrecovery group males or recovery group males or females. Group mean food consumption was 

statistically significantly (p < 0.05) reduced in non-recovery females throughout gestation at 30, 

60, and 120 mg/kg-bw/day, but not during lactation. During gestation days 0–7, group mean 

food consumption was reduced by 12.0, 19.9, and 24.1% at 30, 60, and 120 mg/kg-bw/day, 

respectively, relative to the control group. During gestation days 7–14, group mean food 

consumption was reduced by 11.9, 19.0, and 24.8% at 30, 60, and 120 mg/kg-bw/day, 

respectively, relative to the control group. During gestation days 14–20, group mean food 

consumption was reduced by 12.2, 16.3, and 18.6% at 30, 60, and 120 mg/kg-bw/day, 

respectively, relative to the control group.

Water consumption did not appear to be affected by treatment in non-recovery or recovery group 

animals. “Females treated with 30 mg/kg-bw/day showed a statistically significant [p < 0.05] 

reduction in water consumption during the final week of gestation [11.0% reduction relative to 

the control group]. In the absence of a true dose related response the intergroup difference was 

considered not to be of toxicological importance.” [Dunster 2013, p. 41]
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Trends in food efficiency were similar to those observed in body weight gain. Since no statistics 

were conducted on food efficiency, effects of treatment were not clearly discernible. 

Neurological Assessments:

Behavioral assessments, functional performance tests, and sensory reactivity tests showed no 

treatment-related effects. 

“Females from all [non-recovery] treatment groups showed a statistically significant increase in 

forelimb grip strength when compared to controls. The significance achieved was minimal (p <

0.05) and only occurred during one out of three tests. In the absence of a dose-related response, 

these increases were considered to be unrelated to treatment.” [Dunster 2013, p. 40]

“Females [in the non-recovery group] treated with 120 mg/kg-bw/day showed an increase in 

hind limb grip strength when compared to controls. This was only observed in one out of three 

tests performed, and as such, was considered not to represent a true effect of treatment.” [Dunster 

2013, p. 40]

Hematology and Clinical Chemistry Analyses:

Non-recovery groups:

Group mean platelet count was statistically significantly (p < 0.05) increased in non-recovery

group females at 120 mg/kg-bw/day, with an increase of 16.7%, relative to the control group. 

No other statistically significant effects were observed on hematological parameters in males or 

females of non-recovery groups.

Clinical chemistry evaluations of plasma collected from non-recovery group males showed a

statistically significant (p < 0.05) reduction in the mean urea levels at 120 mg/kg-bw/day, with a 

reduction of 11.6% relative to the control group. In addition, the albumin/globulin ratio was 

statistically significantly (p < 0.05) reduced in non-recovery group males at 120 mg/kg-bw/day, 

with a reduction of 11.7% relative to the control group. “Non-recovery males treated with 30 

mg/kg-bw/day showed a statistically significant [p < 0.05] increase in alkaline phosphatase 

values [of 40.1%] when compared to controls, with one value outside of the normally expected 

ranges. In the absence of a dose-related response, this increase was considered to have arisen 

incidentally and was of no toxicological importance.” [Dunster 2013, p. 43] 

In non-recovery group females, bilirubin was statistically significantly (p < 0.05) increased (by 

33.3%) at 120 mg/kg-bw/day, relative to the control group. In non-recovery females, bile acids 

were statistically significantly (p < 0.05) reduced (by 56.8%) at 120 mg/kg-bw/day, relative to 

the control group. 

Recovery groups:

For hematological parameters, similar to non-recovery females, recovery females showed a 

statistically significant (p < 0.05) increase in the group mean platelet count at 120 mg/kg-bw/day, 

with an increase of 38.4%, relative to the control group. However, the mean platelet count of the 

recovery control group was below the range expected for animals of the same strain and similar 

age, possibly due to one extremely low individual control value (2 x 109/L compared to four 
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other control values ≥ 563 x 109/L). The study report provided no explanation for the low 

individual control value. No other statistically significant effects were observed on 

hematological parameters in males or females of recovery groups.

For clinical chemistry parameters, recovery males exposed to the test substance at 120 mg/kgbw/day showed a statistically significant (p < 0.01) reduction in aspartate aminotransferase 

(reduced by 45.0%, relative to the control group) and a statistically significant (p < 0.05) increase 

in alkaline phosphatase (increased by 35.0%, relative to the control group) at the end of the 2-

week recovery period.

Recovery females exposed to the test substance at 120 mg/kg-bw/day showed a statistically 

significant (p < 0.01) reduction in the albumin/globulin ratio (reduction of 16.1% relative to the 

control group), and a statistically significant (p < 0.05) reduction in alkaline phosphatase 

(reduction of 26.6% relative to the control group) at the end of the 2-week recovery period.

Changes in some of the clinical chemistry parameters examined in this study were observed in 

recovery animals at the end of the 2-week recovery period, but were not observed in nonrecovery animals at the end of the treatment period. These changes included a reduction in 

aspartate aminotransferase in males, an increase in alkaline phosphatase in males, a reduction in 

the albumin/globulin ratio in females, and a reduction in alkaline phosphatase in females. The 

relevance of these changes to test item exposure is not clear and the study design did not permit a 

determination of whether these were delayed responses or were incidental findings. 

Urinalysis:

No treatment-related effects on urinalysis parameters were found in non-recovery or recovery 

males. Urinalysis was not conducted for females.

Gross Necropsy and Histopathology:

No macroscopic findings considered to be related to the treatment were found during necropsy of 

non-recovery or recovery animals. “One male treated with 30 mg/kg-bw/day displayed a fibrous 

mass on the mammary gland. One female treated with 30 mg/kg-bw/day displayed a distended 

left horn of the uterus and a cream colored fluid was present in the vagina. One male treated 

with 120 mg/kg-bw/day displayed small seminal vesicles. Another male treated with 120 mg/kgbw/day showed a yellow fluid-filled mass attached to the left cauda of the epididymis. One 

female treated with 120 mg/kg-bw/day showed reddened eyes. These macroscopic changes were 

considered to be low incidence findings in rats of the age and strain employed, and considered 

not to represent systemic toxicity.” [Dunster 2013, pp. 44–45]

Statistically significant changes in group mean organ weights were observed in non-recovery 

males and females, but not in recovery animals. In non-recovery males, absolute kidney weights 

were statistically significantly (p < 0.05) increased by 12.0%, 8.9%, and 19.6% at 30, 60, and 

120 mg/kg-bw/day, respectively, relative to the control group. Relative kidney weights in nonrecovery males were statistically significantly (p < 0.05) increased by 15.1%, 14.8%, and 25.7% 

at 30, 60, and 120 mg/kg-bw/day, respectively, relative to the control group. Absolute and 

relative liver weights in non-recovery males were statistically significantly (p < 0.01) increased 

by 5.8% and 11.3%, respectively, at 120 mg/kg-bw/day, relative to the control group. Absolute 
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and relative prostate weights in non-recovery males were statistically significantly (p < 0.05) 

decreased by 15.0% and 13.1%, respectively, at 120 mg/kg-bw/day, relative to the control group. 

In non-recovery females, absolute thymus weights were statistically significantly (p < 0.05) 

decreased by 17.5% and 30.6% at 60 and 120 mg/kg-bw/day, respectively, relative to the control 

group. Relative thymus weights were statistically significantly (p < 0.05) decreased by 2.1% and 

17.5% at 60 and 120 mg/kg-bw/day, respectively, relative to the control group. In non-recovery 

females, absolute and relative pituitary weights were statistically significantly (p < 0.05) 

decreased by 18.1% and 4.8%, respectively, at 120 mg/kg-bw/day, relative to the control group. 

No treatment-related histopathological changes were observed during necropsy of non-recovery 

animals. “All of the histopathological findings encountered were considered to have occurred 

spontaneously or at post mortem.” [Dunster 2013, p. 45] Animals from the recovery groups were 

not examined for histopathology.

Determination of LOAEL for Systemic Toxicity:

The only statistically significant changes that were observed in low- and mid-dose males (30 and 

60 mg/kg-bw/day, respectively) were increased mean absolute and relative kidney weights. In 

the absence of corroborating evidence of kidney toxicity from clinical chemistry and/or 

histopathological evaluations, the changes in kidney weight at 30 and 60 mg/kg-bw/day are of 

questionable toxicological significance. Taken alone, the increases in absolute and relative 

kidney weight are insufficient evidence of toxicity in males at 30 and 60 mg/kg-bw/day. 

Statistically significant changes in non-recovery high-dose males (120 mg/kg-bw/day) included

changes in clinical chemistry (reduced mean urea and reduced mean albumin/globulin ratio) and 

organ weight (increased mean absolute and relative kidney and liver weights and reduced mean 

absolute and relative prostate weights); the changes in clinical chemistry, in combination with 

the changes in kidney and liver weights, are suggestive of toxicity to the kidney and/or liver at 

120 mg/kg-bw/day. The toxicological significance of the reduction in absolute and relative 

prostate weights in males at 120 mg/kg-bw/day is unclear, given the absence of corroborating 

evidence of toxicity from histopathological evaluations. For recovery group males exposed to 

120 mg/kg-bw/day and allowed a 2-week recovery period without treatment, statistically 

significant changes in clinical chemistry consisted of reduced mean aspartate aminotransferase 

and increased mean alkaline phosphatase; taken alone, the toxicological relevance of these 

changes in clinical chemistry parameters in recovery males is not clear. Therefore, the high dose 

(120 mg/kg-bw/day) is considered the LOAEL for males based on reduced mean urea, reduced 

mean albumin/globulin ratio, and increased mean absolute and relative kidney and liver weights.

The LOAEL for females is considered to be 30 mg/kg-bw/day, the lowest dose tested, based on 

statistically significantly reduced mean body weight during gestation and lactation, reduced 

mean body weight gain during the pre-pairing period, and reduced mean food consumption

during gestation.

Reproductive and Developmental Toxicity: 

Mating, Fertility, Gestation, and Parturition: No treatment-related changes in the mean pre-coital 

interval, mating index, mean number of corpora lutea, number of implantation sites, pre- and 

post-implantation losses, pregnancy index, parturition index, or gestation length were reported. 

“All animals mated within five days of pairing.” [Dunster 2013, p. 41] 
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Two females treated with 30 mg/kg-bw/day, one female treated with 60 mg/kg-bw/day, and two 

females treated with 120 mg/kg-bw/day failed to become pregnant after evidence of mating. All 

contol females became pregnant. At 0, 30, 60, and 120 mg/kg-bw/day, the pregnancy indices 

were 100%, 83%, 92%, and 83%, respectively. Although the pregnancy index was reduced by 

8–17% in treated animals, a dose-related trend was not evident. 

In addition to the females that did not become pregnant, “[o]ne female treated with 120 mg/kgbw/day showed both corpora lutea and implantation sites, but did not produce a litter. 

Furthermore, there were two total litter losses observed at 120 mg/kg-bw/day and one total litter 

loss observed at 30 mg/kg-bw/day.” [Dunster 2013, p. 42] Females described as having total 

litter losses gave birth to 1 or 2 pups, but none of these pups survived to lactation day 5.

“In total, twelve females from the control group, nine females treated with 30 mg/kg-bw/day, 

eleven females treated with 60 mg/kg-bw/day, and seven females treated with 120 mg/kg-bw/day 

[became pregnant,] gave birth to a live litter and successfully reared young to day 5 of age.” 

[Dunster 2013, p. 42]

Gross Necropsy and Histopathology of Reproductive Organs: No macroscopic findings 

considered to be related to the treatment were found in reproductive organs during necropsy of 

non-recovery or recovery animals. 

As described above, absolute and relative prostate weights in non-recovery males were reduced 

at 120 mg/kg-bw/day, relative to the control group. Absolute and relative prostate weights in 

recovery males were comparable to those of the control group.

No histopathological changes were observed in reproductive organs during necropsy of nonrecovery animals.

Examination of Offspring: No clinical signs of toxicity were observed in the offspring of treated 

animals. “No treatment-related macroscopic abnormalities were detected in interim death or 

terminal kill offspring. The incidental findings observed were those occasionally observed in 

reproductive studies of this type and were considered to be unrelated to toxicity of the test item.” 

[Dunster 2013, p. 44]

No statistically significant differences in group mean values were reported for litter size, litter 

weight, sex ratio, offspring body weight (on lactation day 1 or 4), offspring body weight change 

(lactation day 1–4), live birth index, viability index, or offspring surface righting reflex.

Determination of LOAEL for Reproductive/Developmental Toxicity:

The total number of females that successfully became pregnant, gave birth, and reared young 

until lactation day 5 were 12, 9, 11, and 7 in the control, 30, 60, and 120 mg/kg-bw/day groups, 

respectively; the incidence of 7 out of 12 females at the high dose resulted from 2 females that 

did not become pregnant, 1 female that became pregnant but did not deliver, and 2 females with 

total litter losses. The LOAEL for reproductive and developmental toxicity is considered to be 
Final HPV3 Test Rule Data Adequacy Review (February 2014) CASRN 22527-63-5

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120 mg/kg-bw/day based on the combination of 2 total litter losses and an overall reduction in 

the number of females that reared young until lactation day 5 at this dose. 

CONCLUSIONS

Systemic Toxicity:

The LOAEL for systemic toxicity in females is 30 mg/kg-bw/day, the lowest dose tested, based 

on reduced mean body weight during gestation and lactation, reduced mean body weight gain 

during the pre-pairing period, and reduced mean food consumption during gestation. The 

NOAEL for systemic toxicity in females is not established.

The LOAEL for systemic toxicity in males is 120 mg/kg-bw/day, based on effects on clinical 

chemistry (reduced mean urea and reduced mean albumin/globulin ratio) and increased mean 

absolute and relative kidney and liver weights. The NOAEL for systemic toxicity in males is 60 

mg/kg-bw/day.

Reproductive Toxicity:

The LOAEL for reproductive toxicity is 120 mg/kg-bw/day, based on an increase in total litter 

loss and a reduced number of litters reared until lactation day 5. The NOAEL for reproductive 

toxicity is 60 mg/kg-bw/day.

Developmental Toxicity: 

The LOAEL for developmental toxicity in males and females is 120 mg/kg-bw/day, based on an 

increase in total litter loss and a reduced number of litters reared until lactation day 5. The 

NOAEL for developmental toxicity is 60 mg/kg-bw/day. 

STUDY RELIABILITY

[2] Reliable with restrictions. Calculations of group means and statistical comparisons among 

groups sometimes excluded values, including females that did not become pregnant, females that 

became pregnant but did not deliver a litter, and females with total litter loss. This strategy of 

conducting statistical analyses may have obscured the evidence of a treatment-related effect on 

implantation loss, litter size, offspring viability, and other parameters. 

REFERENCES

Dunster, J. 2013. Benzoflex 1046: Oral (gavage) combined repeat dose toxicity study with 

reproduction/developmental toxicity screening test in the rat (OECD 422 with recovery groups). 

Harlan Laboratories Ltd., Shardlow, Derbyshire, UK, Study No. 41204605. Sponsor: Eastman 

Chemical Company, Kingsport, TN. Study report not available online as of February 18, 2014.
Final HPV3 Test Rule Data Adequacy Review (February 2014) CASRN 22527-63-5

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E. DATA ADEQUACY SUMMARY TABLE 

Endpoint Study Information 

Available

Acceptable Data Gap

Aquatic Effects

Acute Toxicity to Fish No – –

1

Acute Toxicity to Aquatic Invertebrates No – –

1

Chronic Toxicity to Aquatic 

Invertebrates

Yes2 No Yes

Toxicity to Aquatic Plants Yes2 No Yes

Health Effects

Acute Toxicity Yes3 Yes No

Repeated-Dose Toxicity Yes Yes No

Gene Mutation Yes3 Yes No

Chromosomal Aberrations Yes2 Yes No

Reproductive Toxicity Yes Yes No

Developmental Toxicity Yes Yes No

1 Acute toxicity to fish and acute toxicity to aquatic invertebrate testing are not required because 

the measured log Kow of CASRN 22527-63-5 is < 4.2.

2 Data for these endpoints were reviewed in SRC TR-13-060.

3 Data for these endpoints were reviewed in SRC TR-13-050.