SRC TR

- 1 4

- 004

HPV3 TEST RULE DATA ADEQUACY R EVIEW (FINAL ): REPEATED

- DOSE, REPRODUCTIVE, AND DEVELOPMENTAL TO XICITY 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 Protec tion 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 , 2014Final HPV 3 Test Rule Data Adequacy Review (February 2014) CAS RN 22527 - 63 - 5

i 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
................................ ................................ ........... 17Final HPV 3 Test Rule Data Adequacy Review (February 2014) CAS RN 22527 - 63 - 5

1 A. SOURCE MATERIAL USED FOR ASSESSMENT This document evaluates a toxicity test for p ropanoic 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, 2 011 ; referred to herein as HPV3; available online at http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2009

- 0112

- 0086 as of February 1 8 , 201 4 ). The HPV 3 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 K ow < 4.2 or chronic toxicity to Daphnia and toxicity to algae testing if log K ow ≥ 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 K ow of CASRN 22527

- 63

- 5 has been experimentally determined to be ≥ 5.4 (based on measured log K ow 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 incl ude chronic toxicity to Daphnia and toxicity to algae testing. The December 17, 2013 submission 2 from Eastman Chemical Company (“Eastman”) contained the following toxicological test report:  “Benzoflex 1046: Oral (Gavage) Combined Repeat Dose Toxicity St udy with Reproduction/Developmental Toxicity Screening Test in the Rat (OECD 422 with Recovery Groups) ”, submitted for the F 1 testing requirement These newly submitted data are evaluated for adequacy herein. No other toxicity data are evaluated in this r eport. 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 availabl e 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 K ow 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 1 8 , 201 4 ). 2 Submitted via email from L.W. Velsor (Eastman) to J. Schaeffer (EPA) on December 18, 2013.Final HPV 3 Test Rule Data Adequacy Review (February 2014) CAS RN 22527 - 63 - 5

2 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 w ith 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/#!documentDeta il;D=EPA

- HQ

- OPPT

2009

- 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

- pentane diol, 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 manufactur e 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 bu lk 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 an alogs 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 submitt ed 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 Submi ssion 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 th e 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 , 201 4 ), 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 de termined to be adequate to satisfy the D and E1 testing requirements promulgated in the HPV3 test rule. In a nother previous submission (dated June 20, 2013; available online at http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2009

- 0112

- 0145 as ofFinal HPV 3 Test Rule Data Adequacy Review (February 2014) CAS RN 22527 - 63 - 5

3 February 18 , 2014), Eastman supplied data from chronic toxicity to Daphnia , toxicity to algae, and chromosomal aberration tests. These data were reviewed in a s eparate 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, t he 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 aberration s , reproductive toxicity, and developmental toxicity endpoints.Final HPV 3 Test Rule Data Adequacy Review (February 2014) CAS RN 22527 - 63 - 5

4 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 develop mental toxicity endpoint: Adequate Study 1: Combined r epeated - 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 re production/developmental toxicity screening test in the rat (OECD 422 with recovery groups) Th is 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% p ropanoic acid, 2

- methyl

- , 3

- (benzoyloxy)

- 2,2,4

- trimethylpentyl ester (CASRN 22527

- 63

- 5), 19.5% 1,3

- penta nediol, 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 c omposition 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 p ropanoic acid, 2

- methyl

- , 3

- (benzoyloxy)

- 2,2,4

- trimethylpentyl ester (CASRN 22527

- 63

- 5), “dibenzoate” was synonymous with 1,3

- pentanediol, 2,2,4

- tri methyl

- 1,3

- dibenzoate (CASRN 68052

- 23

- 3), and “TXIB” was synonymous with 2,2,4

- trimethyl

- 1,3

- pentanediol diisobu t yrate (CASRN 6846

- 50

- 0).Final HPV 3 Test Rule Data Adequacy Review (February 2014) CAS RN 22527 - 63 - 5

5 Te st species: Wistar Han TM :RccHan TM :WIST strain rats ; obtained from Harlan Laboratories UK, Ltd., Blackthorn, Bicester, Oxon, UK; rats were approximately 12 weeks old and weighe d 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 o il BP Exposure duration: Non

- recovery m ales: 42 days; Non

- recovery f emales: 41

– 4 6 days (throughout 14 days of pre

mating, 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 group s : 0, 30, 60, and 120 mg Benzoflex 1046 /kg

bw/day ; Recovery group s : 0 and 120 mg Benzoflex 1046/kg

- bw/day Statistical methods used: “ Where considered appropriate, quantitative data
... [ w ere] 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 Consum ption 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 Su rface 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 pr egnant, those that became pregnant but did not deliver, and/or those that exhibited total litter loss. “Data were analy z ed using the decision tree from the Provantis TM Tables and Statistics Module as detailed as follows: Where appropriate, data transfor mations were performed using the most suitable method. The homogeneity of variance from mean values was analy z ed using Bartlett’s test. Intergroup variance ... [was] assessed using suitable ANOVA [analysis of variance] , or if required, ANCOVA [analysis of c ovariance] with appropriate covari ates
. Any transformed data were analy z ed to find the lowest treatment level that showed a significant effect using Williams ’ t est for parametric data or the Shirley t est for non

- parametric data. If no dose response was f ound but the data show non

- homogeneity of means, the data were ana lyz ed by a stepwise Dunnet t ’s (parametric) or Steel (non

- parametric) test to determine significant difference [s] from the control group. Where the data were unsuitable for these analyses, p air

- wise tests
... [ were ] performedFinal HPV 3 Test Rule Data Adequacy Review (February 2014) CAS RN 22527 - 63 - 5

6 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 pack age. 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
... [ th ese] data... [ w er e] 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 fol lowed 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 Ma nn - 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 analy z ed u sing non - parametric a nalyses.” [ 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 A rachis 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 w e re 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 d osing 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, g roups of r ats (12/sex/group) were orally administered the test item daily via g avage 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 anim als 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 d ays for males or through postpartum day 4 for females ( 41

– 4 6 consecutive days ) , with the exception that females were not treated during parturition.Final HPV 3 Test Rule Data Adequacy Review (February 2014) CAS RN 22527 - 63 - 5

7 Prior to mating, animals were housed as groups or 4 or 5 / sex. During the mating period, animals were pa ired (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 orig inal 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. M ales were euthanized on day 43. F emales were allowed to deliver and rear their pups until postpartum (lactation) day 5, when all females and their pups were euthanized (day 42 to 4 7 )
. Non

- pregnant females were euthanized on day 25 post coitum
. Recovery group s : A dditional groups of rats (5/sex/group) we re administered the test item via gavage at 0 or 120 mg Benzoflex 1046 /kg

- bw/day for 42 days concurrently with the non

- recovery group s . Recovery animals were observed for two weeks beyond the termination of treatment. Recovery animals were housed as grou ps of 5/sex throughout the treatment period and were not mated. All r ecovery 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 toxic ity, ill

- health , and behavioral change s 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 o f non - recovery animals were recorded on day 1 (prior to dosing), at least weekly throughout the treatment period, and at the time of euthanasia
. I ndividual b ody 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 b ody weights of all r ecovery 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 con sumption was recorded for each cage of [non

recovery] adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for th e periods covering post coitum days 0

– 7, 7

– 14, and 14

– 20. For females with live litters, fo od consumption was recorded on d ays 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 wer e 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 re covery 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. ” [Dunst er 2013, p. 29]Final HPV 3 Test Rule Data Adequacy Review (February 2014) CAS RN 22527 - 63 - 5

8 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 p erformance 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 stren gth. 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 an d 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 th at 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 thrombop lastin time. The following clinical chemistry paramete rs 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 phosp hatase, 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 fin al 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 o rgan weights were recorded for 5 adult animals/sex/group f rom each non

- recovery group and f or all recovery group animals for the following organs: adrenals, brain, heart, kidneys, liver, spleen, thymus, and thyroid /parathyroid ( post
- fixation). Absolute o rgan weights were recorded for all non - recovery and recovery animals for the following organs: epididymides, ovaries, pituitary gland (post

fixation), prostat e gland , seminal vesicles, testes, and uterus with cervix. Relative organ weight s were calculated for each animal as the ratio of absolute organ weight to individual body weight.Final HPV 3 Test Rule Data Adequacy Review (February 2014) CAS RN 22527 - 63 - 5

9 The following tissues collected from 5 non - recovery rats/sex from th e con trol 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, he art, 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

- tho racic, 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 b irth 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 w ere 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 post partum day 0), t he number s of live and dead offspring , and the sex of the offspring, w ere recorded. All live offspring were evaluated for surface righting reflex on postpartum day 1. The following parameters were recorded for each litter during the pos tpartum 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 ti ssues, as described above
. The uterus of each non

- recovery female was examined for signs of implantation . The number s of implantations in each uterine horn and corpora lutea were recorded and used to calculate the pre - and post
- implantation loss. “ Det ailed 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, s ystemic 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 , andFinal HPV 3 Test Rule Data Adequacy Review (February 2014) CAS RN 22527 - 63 - 5

10 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 tox icity) = 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 m g/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 remar ks: 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 occurre d and no treatment

- related clinical signs were observed in adult non recovery 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 in stances 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, i n isolation, is co nsidered not to be indicative of systemic toxicity.” [ Dunster 2013, p. 39 ] Body Weight: Non

- recovery group s : At all doses, f

emale g roup 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 w eights 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. N o significant differences were observed in the body weights of females during th e 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%, resp ectively, at these doses relative to the control group ). Female body weight gains wereFinal HPV 3 Test Rule Data Adequacy Review (February 2014) CAS RN 22527 - 63 - 5

11 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 the se doses relative to the control group ). Female c umulative body weight change during gestation day s 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 group s : No statistically significant differences in body weigh t were observed in males or females of recovery groups. Group mean body weight gain was statistically significantly (p < 0.0 5 ) reduced in recovery group males at 120 mg/kg

- bw/day during the first , second, and final week of the study , compared to control m ales ( reductions of 90.8% , 37.3%, and 137.5% were observed during days 1

– 8, 8

– 15, and 50

– 57, respectiv el y ). Since no statistically significant reductions were observed in the body weight of males in recovery and non

- recovery groups and no statistically si gnificant r eductions 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 we ight ( group mean loss of 2.4 g v ersus 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 statist ically significant (p < 0.05) . Group mean body weight gain in recovery females treated with 120 mg/kg

- bw/day was comparable to the c ontrol group for the remainder of the treatment period and the recovery period
. Food/Water Consumption and Food Efficienc y: Group mean food consumption did not appear to be affected by treatment in exposed non recovery group males or recovery group males or females. Group mean food consumption was statistically significantly (p < 0.05) reduced in non

- recovery females throug hout gestation at 30, 60, and 120 mg/kg

- bw/day, but not during lactation. During gestation day s 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 ges tation day s 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 day s 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
. W ater 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]Final HPV 3 Test Rule Data Adequacy Review (February 2014) CAS RN 22527 - 63 - 5

12 T rends 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: B ehavioral assessmen ts, 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 si gnificance 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 grou p] 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 test s 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 group s : Group m ean 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 gr oup. 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, t he albumin/globulin ratio was statistically significantly (p < 0.05) reduced in non - recovery group males at 120 mg/kg

- bw/d ay, 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 outsid e 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 statistic ally significantly (p < 0.05) increased ( by 33.3%) at 120 mg/kg

- bw/day, relative to the control group. In non

- recovery females, bile acid s were statistically significantly (p < 0.05) reduced ( by 56.8%) at 120 mg/kg

- bw/day, relative to the control group. Recovery group s : For hematological parameters, s imilar 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 contr ol 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 10 9 /L compared to fourFinal HPV 3 Test Rule Data Adequacy Review (February 2014) CAS RN 22527 - 63 - 5

13 other control valu es ≥ 563 x 10 9 /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 paramet ers, r ecovery males exposed to the test substance at 120 mg/kg

bw/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) increas e in alkaline phosphatase (increased by 35.0%, relative to the control group) at the end of the 2

- week recovery period. R ecovery females exposed to the test substance at 120 mg/kg

- bw/day showed a statistically significant (p < 0.01) reduction in the album in/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 non recovery animals at the end of the treatment period. These cha nges included a reduction i n 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 clea r 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 u rinaly sis parameters were found in non

- recovery or recovery males. Urinalysis was no t 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 treate d with 120 mg/kg

bw/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 a ge and strain employed, and considered not to represent systemic toxicity.” [Dunster 2013, pp. 44

– 45] Statistically significant c hanges in g roup mean organ weight s were observed in non - recovery males and females, but not in recovery animals. In non

- recov ery males, a bsolute 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. R elative kidney weights in non

recovery males were statistical ly 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. A bsolute 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. AbsoluteFinal HPV 3 Test Rule Data Adequacy Review (February 2014) CAS RN 22527 - 63 - 5

14 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 wer e 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. I n non - recovery females, absolute and relative pituitary weights were statistically significantly (p < 0.05) decr eased by 18.1% and 4.8 %, respectively, at 120 mg/kg

- bw/day, relative to the control group. No treatment
- related histopathol o gical cha nges were observed during necropsy of non

- recovery animals . “All of the histopathological findings encountered were con sidered 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 obs erv ed 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. S tatistically 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 re duced 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 o f 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 , st atistically significant changes in clinical chemistry consisted of reduced mean aspartate aminotransferase and increased mean alkaline phosphatase ; t aken alone , the toxicolog ical 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 an d 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]Final HPV 3 Test Rule Data Adequacy Review (February 2014) CAS RN 22527 - 63 - 5

15 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%, r espectively. 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/kg

bw/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 g ave birth to 1 or 2 pups, but none of the se 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/k g - 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, a bsolute and relative prostate weights in non

- recovery males were reduced at 120 mg/kg

- bw/day , relative to the control group. A bsolute and relative p rostate weights in recovery males were comparable to those of the control group. No histopathological changes were observed in reproductive organs during necropsy of non

recovery animals. Examination of Offspring: No clinical signs of toxicity were obse rved 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 an d 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, l itter 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 ; th e 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 beFinal HPV 3 Test Rule Data Adequacy Review (February 2014) CAS RN 22527 - 63 - 5

16 120 mg/kg

- bw/day based on the combination of 2 total litter losses and an overall reduction in the numb er 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, r educed mean body weight gain during the pre - pairing period , and reduced mean food consumption during gestation . The NOAEL for systemic toxicity in females i s not establish ed. 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 reprodu ctive 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 toxic ity 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 restricti ons. 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 strat egy of conducting statistical analyses may have obscured the evidence of a treatment

- related effect on implantation loss , litter size, offspring viability , and other parameters. REFERENCE S Dunster, J. 2013
. Benzoflex 1046: Oral ( g avage) c ombined r epea t d ose t oxicity s tudy with r eproduction/ d evelopmental t oxicity s creening t est in the r at (OECD 422 with r ecovery g roups)
. Harlan Laboratories Ltd. , Shardlow, Derbyshire, UK, Study No. 41204605. Sponsor: Eastman Chemical Company, Kingsport, TN. Study repo rt not available online as of February 18 , 2014.Final HPV 3 Test Rule Data Adequacy Review (February 2014) CAS RN 22527 - 63 - 5

17 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 Toxici ty to Aquatic Invertebrates Yes 2 No Yes Toxicity to Aquatic Plants Yes 2 No Yes Health Effects

Acute Toxicity Yes 3 Yes No Repeated

- Dose Toxicity Yes Yes No Gene Mutation Yes 3 Yes No Chromosomal Aberrations Yes 2 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 K ow 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.