SRC TR

- 13

- 071

HPV 2 TEST RULE DATA ADEQUACY REVIEW (FINAL ) : REPEATED

- DOSE

, REPRODUCTI VE, AND DEVELOPMENTAL TOXICI TY

D

- gluco

- heptonic acid, monosodium salt, (2.xi.)

- , CASRN 31138

- 65

- 5

Prepared by:

Ashlee Aldridge, Amy Nguyen, Teresa Manyin, and William 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 .009.001

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

November 1

, 2013Final HPV 2 Test Rule Data Adequacy Review (November 2013) CAS RN 31138

- 65

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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

................................ ................................ .............................. 3

D

- 1. Repeated

- Dose, Reproductive, and Developmental Toxicity

................................ ............ 3
E. DATA ADEQUACY SUMMARY TABLE

................................ ................................ ........... 15Final HPV 2 Test Rule Data Adequacy Review (November 2013) CAS RN 31138

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- 5

1 A. SOURCE MATERIAL USED FOR ASSESSMENT

This document evaluates a toxicity test for D

- gluco

- heptonic acid, monosodium salt, (2.xi.)

- (CASRN 31138

- 65

- 5) submitted by Harcros Chemicals, Inc. ( on August 27 , 2013 ) to EPA in response to the final test rule, “Testing of Certain High Production Volume Chemicals; Second Group of Chemicals” (76 FR 1067, January 7, 2011; referred to herein as HPV2 ; available online
at http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0579 as of November 1 , 2013 ).

T he HPV2 test rule requires testing of CASRN 31138

- 65

- 5 for the following toxic ological endpoints: 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 t est), and F1 (combined repeated

- dose toxicity study with the reproduction/developmental toxicity screening test). Since the log K ow of CASRN 31138

- 65

- 5 is estimated to be

- 6.44 1 , which is < 4.2, the C1 aquatic toxicity testing requirement is considered to include acute toxicity to fish, acute toxicity to Daphnia , and toxicity to algae testing.

The August 27, 2013 submission from Harcros Chemicals, Inc. included the following:

 Oral (Gavage) Combined Repeat Dose Toxicity Study with Reproduction/Developmental Toxicity Screening Test in the Rat

, submitted for F 1 testing requirement ( submitted via email from A.M. Savini [Harcros Chemicals, Inc.] to J. Schaeffer [EPA]; submission not available online as of November 1 , 2013 )

.

Th ese newly submitted data are evaluated for adequacy herein. No other toxicity data are evaluated in this report.

B. EVALUATING DATA ADEQUACY

The newly submitted data 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 these data were adequate to satisfy SIDS endpoints for repeated

- d ose, reproductive, and developmental toxicity , study details were compared to current standard test guidelines to establish whether the study met current testing requirements. A table summarizing data availability/adequacy for each SIDS endpoint is includ ed on the last page of this document (Section E).

1 U.S. EPA. ( 2012 ) Estimation Programs Interface SuiteTM (EPI Suite) for Microsoft® Windows, v4.11. U.S. Environmental Protection Agency, Washington, DC, USA. Available online at http://www.epa.gov/opptintr/ exposure/pubs/episuitedl.htm as of November 1 , 2013.Final HPV 2 Test Rule Data Adequacy Review (November 2013) CAS RN 31138

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

Data supplied in the submission identified above are adequate to satisfy the combined repeated

dose toxicity with reproduction/developmental toxicity screening (F1 ) testing requirement promulgated in the final HPV2 test rule.

In six previous submissions ( dated from December 18, 2012 to June 12 , 2013), Harcros Chemicals, Inc. submitted data for acute toxicity to fish (component of C1), acute toxicity to Daphnia (component of C1), toxicity to plants (algae) (component of C1), acute mammalian toxicity (D), gene mutation (E1), and chromosomal aberrations (E2) testing requirements. These submissions included the following:

 Acute Toxicity to Rainbow Trout, submitted for C1 testing requirement on April 24, 2013 (available online at http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

OPPT

- 2007

- 0531

- 07 15 as of November 1 , 2013 )

 Acute Toxicity to Daphnia sp., submitted for C1 testing requirement on June 12, 2013 (available online at http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

2007

- 0531

- 0719 as of November 1 , 2013)

 Algal Inhibition Test, submitted for C1 testing requirement on April 23, 2013 (available online at http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0716 as of November 1 , 2013 )

 Acute Toxicity in the Rat

– Up and Down Procedure, submitted for D testing requirement on March 19, 2013 (available online at http://www.regulations .gov/#!documentDetail; D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0710 as of November 1 , 2013 )

 Reverse Mutation Assay “Ames test” using Salmonella typhimurium and Escherichia coli , submitted for E1 testing requirement on December 18, 2012 (available online at http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0688 as of November 1 , 2013) 1

 Micronucleus Test in the Mouse, submitted for E2 testing requireme nt on March 7, 2013 (available online at http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

2007

- 0531

- 0709 as of November 1 , 2013 )

The data from the submi ssions above have been reviewed in separate document s (SRC Technical Report s # 1 3

- 045 and # 13

- 059 ). The data were determined to be adequate to satisfy the toxicity to plants (algae) ( component of C 1) , acute mammalian toxicity (D), gene mutation (E1)

, and chromosomal aberrations (E2) test ing requirement s, but not adequate to satisfy the acute toxicity to fish (component of C 1) or acute toxicity to Daphnia (component of C1) testing requirement s

. Summary of Data Adequacy for Toxicological Endpoints: Data submitted by Harcros Chemicals, Inc. for the acute toxicity to fish and acute toxicity to Daphnia endpoints are not considered adequate; these tests need to be repeated without pH adjustment of test solutions. Adequate data have been submitted by Har cros Chemicals, Inc. for toxicity to plants (algae), acute mammalian toxicity, repeated

- dose toxicity, gene mutation

, chromosomal aberrations , reproductive toxicity, and developmental toxicity endpoints.

1 Harcros Chemical, Inc. submitted an amendment to this study report on May 22, 2013 (available online at http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0718 as of November 1 , 2013) that indicated that formulated concentrations used in Experiments 1 and 2 were adjusted for water content (49.5%).Final HPV 2 Test Rule Data Adequacy Review (November 2013) CAS RN 31138

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3 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 r epeated

- dose oral toxicity study with reproduction/developmental toxicity screening test in rats (

McRae and Watson , 2013 )

Title: Sodium Glucoheptonate (EC 250

- 480

- 2): Oral (Gavage) Combined Repeat Dose Toxicity Study w ith Reproduction/Developmental Toxicity Screening Test i n t he Rat

Th is study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: D

- gluco

- heptonic acid, monosodium salt, (2.xi.)

- (synonym: sodium glucoheptonate) ; CASRN 31138

- 65

- 5

Remarks: Dark brown liquid ; Batch No. 921000100; purity reported as 50.5% (with 49.5% water) in study report; however, in an email from A.M. Savini (Harcros Chemicals, Inc.) to J. Schaeffer (EPA) on March 5, 2013, the composition of the test substance was reported as follows: 23.4% Beta Sodium Glucoheptonate (CASRN 30090

- 50

- 3 1 ) 20.62% Alpha Sodium Glucoheptonate (CASRN 13007

- 85

- 7) 6.88% Polysacchari des or Degradation Products 2.84 ppm Cyanide 1038 ppm Ammonia 49.1%Water pH = 9.62 Specific Gravity = 1.28 % Solids = 50.9% No dextrose detected The above composition is identical to that provided by Harcros Chemicals, Inc. in its proposal for testing, available at online at http://www.regulations.gov/#!do cumentDetail;D=EPA

- HQ

OPPT

- 2007

- 0531

- 0639 as of November 1 , 2013. Based on this information, the purity of the test substance was 44.02% a.i. 2 (23.4% beta sodium glucoheptonate + 20.62% alpha sodium glucoheptonate).

1 30090

- 50

- 3 is not a valid CAS number according to the U.S. National Library of Medicine’s ChemIDplus website ( http://chem.sis.nlm.nih.gov/chemidplus/ ) ; however, based on the chemical name provided (beta sodium glucoheptonate), this component is assumed to be a stereoisomer of CASRN 31138

- 65

- 5

. 2 Active ingredient (a.i.) = CASRN 31138

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- 5Final HPV 2 Test Rule Data Adequacy Review (November 2013) CAS RN 31138

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4 METHOD

Method/guideline followed: OECD TG 422 (1996) “Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test" ; Commission Regulation (EC) No. 440/2008

Test type: Combined Repeat ed

- Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

GLP compliant?: Yes

Year study performed: 2013

Test species: Wistar Han TM : RccHan TM :WIST strain rats from Harlan Laboratories UK Ltd., Oxon, UK; at initiation, rats were a pproximately 12 weeks old and weighed 302

- 346 grams ( males ) and 191

- 217 grams ( females )

Number of animals/sex/dose: 12

Route of administration: Oral ( gavage )

Vehicle: Distilled water

Exposure duration: Males: 42 days; Females: 41

- 45 days ( throughout 14 days of pre

- mating, mating [maximum of 14 days ]
, g estation, and up to post

- partum/lactation day 4 )

Doses/concentrations tested: Nominal doses: 0, 59.4

, 594

, and 1980 mg test item /kg

- bw/day ; Doses adjusted for % active ingredient ( based on 44.02% purity): 0 (control), 26.1, 261 , and 872
mg a.i. /kg

- bw /day

. 1

Statistical methods used:

“ Where considered appropriate, quantitative data was 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 [ analys e s w ere] performed on the following parameters: Grip Strength, Motor Activity, Body Weight, Body Weight Change, Food 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, Haematology, Blood Chemistry, Absolute Organ Weights, Body Weight

- Relative Organ Weights. ” [ McRae and Watson 2013, p. 32]

1 The study author reported nominal doses of 0, 30, 300, and 1000 mg/kg

- bw/day , which were calculated by adjusting for a purity of 50.5% (water content of 49.5%). The correct test substance purity was 44.02%. The concentrations presented in the text above have been corrected for 44.02% purity.Final HPV 2 Test Rule Data Adequacy Review (November 2013) CAS RN 31138

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5 For litter responses: “ The standard unit of assessment was considered to be the litter, therefore, values were first calculated for each litter and the group mean was calculated using the individual litter values. Group mean values included all litters reared to termination (Day 5 of age). ” [ McRae and Watson 2013, p. 31]

“ Data were analyzed using the decision tree from the ProvantisTM Tables and Statistics Module as detailed below: 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 variances were 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 the Williams Test for parametric data or the Shirley Test for non

- parametric data. If no dose response was found, b ut the data showed non

- homogeneity of means, the data were analyz ed by a stepwise Dunnett’s (parametric) or Steel (non

- parametric) test to determine [if there were any] significant difference [s] from the control group. Where the data were unsuitable for th ese analyses, pair

wise tests [were] performed using the Student [’ s] t

- test (parametric) or the Mann

- Whitney U test (non

- parametric). ” [ McRae and Watson 2013, pp. 32

- 33]

“ Data not analyzed by the Provantis data capture system were assessed separately usin g 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 (ANOV A). If this data was 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 analyz ed incorporating the Kruskal

- Wallis test which if s ignificant, was followed by the Mann

- Whitney U test. Dose response relationship [s] [were] investigated by linear regression. Where the data [were] unsuitable for these analyses, then pair

- wise tests were performed using the Student t

- test (parametric) or the Mann

- Whitney U test (non

- parametric). ” [ McRae and Watson 2013, p. 33]

“ Due to the preponderance of non

- normally distributed data, reproductive parameters (implantation losses, offspring sex ratio and offspring surface righting) were analyzed using non

parametric analyses. ” [ McRae and Watson 2013, p

. 33]

Test method/conditions remarks: Analyses of Test Item Formulations: Based on s tability tests showing prepared bulk formulations to be stable for at least 7 days ( test data not provided), bulk formulations were prepared on a weekly basis and stored at approximately 4 °C in the dark . Homogeneity of form ulations was assessed visually. During the study, samples of the test item formulations (nominal concentrations of 0, 1 1.9, 118.8, and 396
mg test item

/mL , corresponding to doses of 0, 26 .1 , 26 1 , and 872 mg a.i. /kg

- bw/day, respectively ) were collected within 4 days of preparation and analyzed by high performance liquid chromatography mass spectrometry (HPLC

- MS ) to verify exposure concentrations

.Final HPV 2 Test Rule Data Adequacy Review (November 2013) CAS RN 31138

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6 Dosing and Environmental Conditions: Groups of rats (12/sex/group) were orally administered the test item daily via gavage at dose levels of 59.4, 594, and 1980 mg test item/kg

- bw/day ( 26 .1 , 26 1 , and 872 mg a.i. /kg

- bw/day

, respectively) at a constant dosing volume of 5 mL/kg

- bw. A concurrent control group was treated with the vehicle only. Treatment continued throughout the pre

- mating (14 days), mating (maximum of 14 days), and post

- mating periods. Exposure continued in m ales and females until scheduled euthanasia, for total exposure periods of 42 consecutive days (males) or 41

- 45 consecutive days (females) with the exception of during parturition.

Prior to mating, rats were housed as groups of 4/sex. F ood and water were available ad libitum throughout the study. During the mating period, animals were paired (1 male: 1 female) until there was evidence of mating (i.e., the presence of sperm in a vaginal smear and/or the presence of a vaginal plug). Fo llowing evidence of mating (designated as day 0 post

- coitum), males were returned to the original cage. Mated females were housed individually (or with their litters after delivery) throughout gestation and lactation.

M ales were euthanized on day 43

. F emales were all owed to deliver and rear their pups until post

- partum (lactation) day 5, when all females and their pups were euthanized. Non

- pregnant females were euthanized on day 25 post

- coitum.

Clinical Observations: All animals were observed for overt signs of toxicity, ill

- health, and behavioral change s immediately before dosing and two to three times after each daily dose.

Body Weight and Food/Water Consumption: Individual body weights of parental males and females were recorded at least weekly and at the ti me of sacrifice. Food consumption was recorded weekly on a per

- cage basis for males during the pre

- mating and post

- mating periods and for females during the pre

- mating period. Food consumption was also recorded for weekly intervals for each female during the post

- mating period (post

- coitum d ays 0

- 7, 7

- 14

, and 14

- 20 ) and between post

- partum days 1 and 4 for females with live litters. Water intakes were measured daily during the first two weeks of the study.

Fo od efficiency (the ratio of body weight change/dietary intake) was retrospectively calculated for males throughout the study (except during mating) and for females during pre

- pairing. “Due to offspring growth and milk production, food efficiency could not be accurately calculated for females during gestation and lactation.” [McRae and Watson 2013, p. 25]

Neurological Assessments: Animals were observed for signs of functional and behavioral toxicity prior to the start of treatment and at weekly intervals thereafter. Functional performance tests and sensory reactivity
assessments were performed on 5 parental rats/sex/group at the completion of p airing (during week 6) in males or on post

- partum day 4 in females

. Functional performance tests evaluated motor activity and forelimb/hindlimb grip strength while sensory reactivity tests measured

r eactivity to auditory, visual, and proprioceptive stimuli. Measured s ensory reactivity parame tersFinal HPV 2 Test Rule Data Adequacy Review (November 2013) CAS RN 31138

- 65

- 5

7 included grasp response, vocalization, toe pinch, touch escape, pupil reflex, pinch reflex, tail pinch, and finger approach.

All animals were evaluated in b ehavioral assessments for the following parameters: gait, tremors, twitches, hyper/hypothermia, skin color, respiration, convulsions, bizarre/abnormal/stereotypic behavior, salivation, pilo

- erection, exophthalmia, lachrymation, palpebral closure, urination, defecation, transfer arousal, and tail elevation.

Hematology and Clinical Chemistry Measurements: Blood samples were collected from 5 parental rats/sex/group on the day prior to sacrifice for blood and serum chemistry analyses.

The following hematological parameters were evaluated: hemoglobin, erythrocyte count ( i.e., red blood cell count , RBC) , hematocrit, erythrocyte indices ( mean corpuscular hemoglobin, mean corpuscular volume, and mean corpuscular hemoglobin concentration ) , total leu k ocyte count ( i.e., white blood cell count , WBC )

, differential leukocyte count s ( including counts for neutrophils, lymphocytes, monocytes, eosinophils, and basophils ) , platelet count, prothrombin time, and activated partial thromboblastin time.

Serum chemistry parameters that were evaluated included al bumin, total protein, albumin/globulin ratio , total bilirubin, urea , creatinine, alkaline phosphatase, alanine aminotransfera se, aspartate aminotransferase, glucose, total cholesterol, calcium, chloride, potassium, sodium, bile acids, and inorganic phosp horus

. Gross Necropsy and Histopathology: All adults were subjected to a full external and internal examination and selected organs were weighed. Organ weights were recorded f or 5 parental rats/sex/group for the adrenals , brain, spleen, heart, kidneys

, liver, thymus

, and thyroid with parathyroids. Additionally, o rgan weights were recorded f

or all animals in all groups for the epididymides, prostate, seminal vesicles, ovaries, pituitary gland, testes, and uterus with cervix.

The following tissues , collected from 5 parental rats /sex from the control and high

- dose groups, were examined by microscopy : all gross lesions, adrenal glands, aorta, bone and bone marrow (femur with stifle joint and sternum), brain (cerebrum, cerebral cortex , and medulla/po ns),

c ecum, colon, duodenum, epididymides, eyes, heart, ileum (with Peyer’s patches), jejunum, kidneys, liver, lungs and bronchi, lymph nodes (cervical and mesenteric), mammary gland, muscle (skeletal), esophagus, ovaries, pancreas, parathyroid gland, pros tate gland, pituitary gland, rectum, salivary gland (submaxillary), sciatic nerve, seminal vesicles with coagulating glands, skin (hindlimb), spinal cord (cervical, lumbar, and thoracic), spleen, stomach, testes, thymus, thyroid glands, trachea, urinary bl adder, uterus, uterine cervix, and vagina.

Histological examinations were also performed on the following tissues from all remaining animals of the control and high

- dose groups

, all infertile males of any dose group, and all females that did not produce a litter: all gross lesions, epididymides, coagulating glands, mammary gland, ovaries, pituitary gland, prostate gland, seminal vesicles, testes, uterus, uterine cervix, and vagina.Final HPV 2 Test Rule Data Adequacy Review (November 2013) CAS RN 31138

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8 Reproductive/Developmental Toxicity: P regnant female s were observed daily and the following parameters were recorded: date of pairing, date of mating, dat e and time of observed start of parturition, and date and time of observed completion of parturition

. Parameters recorded on and after post

- partum day 0 and used to calculate litter responses included number of offspring born, number of live offspring on post

- partum day s 1 and 4, sex of the offspring on post

- partum days 1 and 4, and individual offspring weights on post

- partum days 1 and 4 (individual weights were used to retrospectively calculate litter weights). Calculated r eproductive parameters i ncluded pre

- coital interval, mating index, pregnancy index, gestati on length, parturition index, pre

- implantation loss, post

implantation loss, live birth index, viability index, and sex ratio.

Clinical condition of the o ffspring w as recorded from birth to post

- partum day 5 . Offspring surface righting reflex was evaluated on post

- partum day 1.

At necropsy, reproductive organ weights were recorded for all parental animals. Reproductive organs weighed included the epididymides, prostate, seminal vesicles, ovaries, testes, and uterus with cervix . Histologi cal examinations were performed on selected reproductive tissues collected from all animals of the control and high

- dose group, including the epididymides, ovaries, mammary gland, prostate gland, seminal vesicles , coagulating gland , testes, uterus, cervix, and vagina. The number s of uterine implantations and corpora lutea were recorded for each female upon examination of the uterus.

RESULTS

Endpoint value(s): Systemic Toxicity: L OAEL ( male and female, systemic toxicity) = 1980 mg test item/kg

- bw/day (= 872 mg a.i. /kg

bw/day )

, based on effects on hematology, clinical chemistry, and organ weights. In males, effects included reduced hemoglobin, erythrocyte count, hematocrit, and serum total protein, elevated albumin/globulin ratio, and decreased absolu te and relative pituitary gland weights . In females, effects included reduced serum calcium and inorganic phosphate, reduced total leukocyte and lymphocyte counts, increased eosinophil count, elevated absolute and relative thyroid/parathyroid weights, and decreased absolute and relative combined uterus and cervix weights

. NOAEL (male and female, systemic toxicity) = 594 mg test item/kg

- bw/day (= 261 mg a.i./kg

bw/day).

Reproductive Toxicity: L OAEL ( male and female, reproductive toxicity) = 1980 mg test item/kg

- bw/day (= 872 mg a.i. /kg

- bw/day )

, based on reduced litter size at birth

, decreased absolute and relative combined uterus and cervix weights , and decreased absolute and relative pituitary gland weight in males

. NOAEL (male and female, reproductive toxicity) = 594 mg test item/kg

- bw/day (= 261 mg a.i./kg

- bw/day).Final HPV 2 Test Rule Data Adequacy Review (November 2013) CAS RN 31138

- 65

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9 Developmental Toxicity: L OAEL (male and female, developmental toxicity) = 1980 mg test item/kg

- bw/day (= 872 mg a.i. /kg

- bw/day )

, based on reduced litter size at birth

. NOAEL (male and female, developmental toxicity) = 594 mg test item/kg

- bw/day (= 261 mg a.i./kg

- bw/day).

Results remarks: Analyses of Test Item Formulations: Measurement of test item concentrations by HPLC

- MS in formulations prepared for dosing (nominal concentrations of 11.9, 118.8

, and 396 mg test item /mL, which corresponded to doses of 26 .1, 261 , and 872 mg a.i. /kg

- bw/day, respectively ) showed that formulations contained 89

- 122% of nominal concentrations.

Clinical Observations: No mortalities occurre d and no treatment

- related clinical signs were observed in parental animals

. Incidental findings included noisy respiration developing from day 31 in one female in each of t he 26.1 and 261 mg a.i. /kg

- bw/day dose groups and in one male in the 872 mg a.i./ kg

bw/day dose group

. On day 42, the affected female in the 26.1 mg a.i. /kg

- bw/day dose group also displayed labored respiration, increased salivation, pilo

- erection

, and hunched postur e.

“ Given the isolated nature of these changes, they were considered attributable to the gavage procedure rather than a direct effect of the test item (or vehicle). ” [McRae and Watson 2013, p. 35]

“ Four males treated with [261 mg a.i. /kg

- bw/day ] ha d generaliz ed scab formation from d ay 19 onwards. Observations of this nature are commonly o bserved in group housed animals and result from social interaction and are therefore co nsidered unrelated to test item toxicity. ” [ McRae and Watson 2013, p. 35]

Body Weight and Food/Water Consumption: G roup mean weight gains at week 2 were s tatistically significantly decreased (p < 0.05) in males treated with 872 mg a.i. /kg

- bw/day , relative to controls, but showed recovery thereafter. Group mean b ody weight gain in females t reated with 872 mg a.i. /kg

- bw/day w as slightly lower throughout the study compared with controls. However, the de crease was not statistically significant and a dose

- related trend was not apparent. No adverse effects were observed on food consumption or food efficiency throughout the study or on water consumption during the pre

pairing phase.

Neurological Assessments: No treatment

- related changes were observed for group mean behavioral assessments , sensory reactivity assessments, or functional performance tests

. In functional performance tests

, a statistically significant increase (p < 0.05) , relative to controls, in one of three hindlimb grip strength measurements was observed in females treated with 261 and 872 mg a.i. /kg

- bw/day , but the rele vance of the increase is not clear and the finding does not appear to be toxicologically relevant.Final HPV 2 Test Rule Data Adequacy Review (November 2013) CAS RN 31138

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- 5

10 Hematology and Clinical Chemistry Measurements: S tatistically significant decreases in hemoglobin (p < 0.05), erythrocyte count (p < 0.05), and hematocrit (p < 0.01) w ere observed in males in the 872 mg a.i. /kg

- bw/day group relative to the control group

. High

- dose male mean values for hemoglobin, erythrocyte count , and hematocrit

, respectively, were approximately 1 0.7 %, 9 .1 %, and 11 .3 % less than control means

.

Si gnificant decrease s in the mean total leu k ocyte count (p < 0.05) and mean lymphocyte count (p < 0.05)

, as well as a significant increase in the mean eosinophil count (p < 0.05)

, were noted in females in the high

- dose group relative to controls. The high

- dose female mean total leukocyte and lymphocyte count s were 34 .5 % and 39 .2 % lower than controls, respectively. Eosinophils were detected in two females from the high

- dose group, but in none from the control group. Mean eosinophil counts in fem ales in the control and high

- dose group s were 0 .000 and 0.028 x 10 9 /L, respectively. Although all values for total leukocyte and lymphocyte counts from individual rats were within the historical control ranges, the reductions in these counts taken in combination with the change in eosinophil count (outside historical control range) are sug gestive of a toxicologically relevant effect.

No other statistically significant changes in hematological parameters were observed in males or females

. Significant changes in clinical (serum) chemistry measurements in males included s ignificant reduction s in mean total protein and mean creatinine , and significant increases in the mean albumin/globulin ratio in both the 261 and 872 mg a.i. /kg

- bw/day groups

. Mean total protein was decreased (p < 0.05) by approximately 8 .3 % and 10 .3 % in mid

- and high

- dose group male s, respectively, relative to the control group. Mean creatinine was decreased (p < 0.05) by approximately 16 .1 % and 22 .0 % in mid

- and high

- dose group males, respectively , relative to the control group , but all creatinine measurements for individ ual rats were within the historical control range . The mean albumin/globulin ratio was increased (p < 0.05 and p < 0.01, respectively) by approximately 14.2 % and 19.5 % in mid

- and high

- dose group males, respectively , relative to the control group

.

In females, significant decrease s in mean inorganic phosphate concentrations w ere observed in females in both the mid

- and high

- dose groups , with decreases of 31 .3 % (p < 0.05) and 60 .2 % (p < 0.01) , respectively, relative to the control group ; however, all ind ividual phosphate measurements at the mid

- dose were within the historical control range

. A s ignificant decrease (p < 0.05) in mean calcium w as also observed in females in the high

- dose group ; the decrease was approximately 7.4 % relative to the control gro up. A significant increase (p < 0.05) in mean plasma glucose was observed in females in the low

- dose group , but t he absence of a dose

- related trend or similar effects on mean plasma glucose levels in other dose groups suggests that th e increase in the low

- dose group may be an incidental finding.

No other statistically significant changes in clinical chemistry parameters were observed in males or females

.Final HPV 2 Test Rule Data Adequacy Review (November 2013) CAS RN 31138

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11 Gross Necropsy and Histopathology: One male in the high

- dose group had a dark spleen and reddened lungs . Microscopic examination of this animal showed congestion in both the spleen and lungs and modera te peribronchiolar intra

- a lveolar pink fluid in the lungs . “ This [finding] may have been a result of a partial mal

- dose during the study

.. . [G] iven the isolated nature of this finding

, it was considered incidental and not to be related to test item toxicity. ” [ McRae and Watson 2013, p. 41]

O ne female in the high

- dose group had a mass under the right forelimb at necropsy , which was confirmed t o be an adeno carcinoma. This finding may have been sporadic as no similar findings

were seen in other animals of the high

- dose group or other groups.

Mean a bsolute and relative pituitary weights were statistically significantly decreased (p < 0.01) in males treated with 872 mg a.i. /kg

- bw/day compared to controls , with decreases of approximately 20 .1 % and 33 .3 % for absolute and relative pituitary weights , respectively.

Females in the high

- dose group showed statistically significant increase s (p < 0.05) in mean absolute an d relative thyroid/parathyroid weights, wi th increases of approximately 44.7 % and 40.0 % for absolute and relative thyroid/parathyroid weights , respectively, relative to controls

. Statistically significant de crease s (p < 0.05) in m ean absolute and relative combined uterus and cervix weights (i.e., organ weights for both the uterus and cervix) were found in females in the high

- dose group. In this group, the mean absolute and relative combined uterus and cervix weights were approxima tely 14.3% and 15.3% lower, respectively, than the control group.

N o toxicologically relevant histopathological changes were noted in the test animals relative to the controls. “ All of the histopathological findings encountered were considered to be within the range of normal background lesions which may be recorded in animals of this strain and age.” [ McRae and Watson 2013, p. 476 ]

Determination of LOAEL for Systemic Toxicity : The only statistically significant changes that were observed at the mid

- dose (261 mg a.i. /kg

bw/day ) were reduced total protein, reduced creatinine (within historical control range), and increased albumin/globulin ratio in males and reduced inorganic phos phate in females (within historical control range). Although the changes in total protein and albumin/globulin ratio in mid

- dose males are suggestive of kidney toxicity, in the absence of corroborating evidence from histopathological evaluations, these re latively small changes are of questionable toxicological significance. The changes in creatinine in males and inorganic phosphate in females were within historical control ranges and, therefore, considered incidental to treatment. Taken alone, the protei n and albumin/globulin changes are insufficient evidence of toxicity at the mid

- dose. Therefore, the high dose (872 mg a.i. /kg

- bw/day ) is considered the LOAEL based on effects on hematology, clinical chemistry, and organ weights. In males , effects at the high dose included reduced hemoglobin, erythrocyte count, hematocrit, and serum total protein, elevated albumin/globulin ratio, and decreased absolute and relative pituitary gland weights 1 . In females

,

1 Although no treatment

- related histological changes were observed in the pituitary, the primary rationale for including decreased pituitary gland weight in high

- dose males as one of the determinants of the LOAEL for systemic toxicity is the magnitude of th e decrease (approximately 20.1% and 33.3% for absolute and relative pituitary weights, respectively , compared to controls ).Final HPV 2 Test Rule Data Adequacy Review (November 2013) CAS RN 31138

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12 effects at the high dose included re duced serum calcium and inorganic phosphate, reduced total leukocyte and lymphocyte counts, increased eosinophil count, elevated absolute and relative thyroid/parathyroid weights 1 , and decreased absolute and relative combined uterus and cervix weights 2

.

R eproductive /Developmental Toxicity: In total, 10 control females, all 12 females from the 26.1 mg a.i./kg

- bw/day dose group, 11 females from the 261 mg a.i. /kg

- bw/day dose group

, and all 12 females from the 872 mg a.i. /kg

bw/day dose group gave birth to a live litter a nd successfully reared young to post

- partum d ay 5.

Mating, Fertility, Gestation, and Parturition: No treatment

- related effects were observed on mating, fertility, or gestation parameters. The mean pre

- coital interval, mating index, mean

nu mber of pregnancies, gestation length, number of females with live offspring, and pregnancy and parturition indices were similar among the treated and control groups. The mean number of corpora lutea and number of implantation sites were also similar among treated and control
groups. Although pre

- and post

- implantation losses appeared slightly elevated at the high dose relative to the control, the differences were not statistically significant. Gestatio n lengths ranged from 22 to 241⁄2 days for all animals. “With the exception of one paired control male and female [with no evidence of mating] all females mated within the first four days of pairing.” [McRae and Watson 2013, p. 37]

One control female did not become pregnant following evidence of mating

. One female in the mid

- dose group also did not become pregnant following evidence of mating ; however, no abnormalities were found in the reproductive organs of this female. The male partner of this female was found to have small epididymides at necropsy, with s evere bilateral hypospermia and severe bilateral g erminal epithelial atrophy found upon histopathological examination, which may at least partially explain the lack of pregnancy in this mid

- dose femal e. All other females successfully mated and became pregnant.

Examination of Reproductive Organs: As stated above, statistically s ignificant d ecreases (p < 0.05) in mean absolute and relative combined uterus and cervix weights were observed in females in the high

- dose group. H istopathology revealed n o treatment

- related changes in the uterus or cervix of the high

- dose group or any other group

. As indicated above, o ne male in the mid

- dose group had small epididymides and small flaccid testes . H istopathological examination of this male revealed severe bilateral hypospermia of the epididymides and severe bilateral germinal epithelial atrophy of the testes

. Given the absence of these or other abnormalities in the

1 Although no treatment

- related histological changes were observed in the thyroid/parathyroid, the rationale for including increa sed thyroid/parathyroid weight in high

- dose females as one of the determinants of the LOAEL for systemic toxicity is the magnitude of the increase (approximately 44.7% and 40.0% for absolute and relative thyroid/parathyroid weights, respectively , compared to controls ).

2 Although no treatment

- related histological changes were observed in the uterus/cervix, the decreased absolute and relative weight of the uterus/cervix seen in high

- dose females is included as one of the determinants of the LOAEL for systemi c toxicity because “[d]ecreased or increased uterine weight” is an effect “on the uterus that may be considered adverse” ( http://www.epa.gov/raf/publication s/guidelines

- reproductive

- tox

- risk

- assessment.htm p. 41).Final HPV 2 Test Rule Data Adequacy Review (November 2013) CAS RN 31138

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13 epididymides and testes of any other treated males, the se findings are not clearly related to the
treatment

. Clinical Examinations of Offspring: “ No overt clinical evidence of toxicity was detected for offspring from treated females when compared to controls. The incidental clini cal signs detected throughout the control and treated groups, consisting of small size, offspring found dead or missing, bruising, cold to the touch, and physical injuries

, were considered to be low incidence findings observed in offspring in studies of th is type, and were unrelated to test item toxicity. ” [McRae and Watson 2013, p. 39] No treatment

- related effects were observed on the surface righting reflex of offspring.

Offspring Viability and Growth: Mean litter size at birth was statistically significantly decreased (p < 0.05) in the high

- dose group (range: 3

- 16 offspring; mean: 10.4) , with a decrease of approximately 2 1.8 % relative to the control group (range: 10

- 18 offspring; mean: 13.3)

. In the high

- dose group, 4/12 litters were smaller than the control group, producing only 3, 4, 9, and 9 offspring versus 10 or more offspring produced in l itters from the control group. Differences in

m ean litter size were not statistically significant on post

- partum day s 1 or 4. Mean live birth index, viability index, and sex ratio were similar among the treated and control groups. Total litter loss was observed only for one control female between post

- partum days 1 and 4.

A decrease in mean litter weight on post

- partum day 1 , which was not statistically significant, was noted in the high

- dose group relative to the control group

. The same trend was observed on post

- partum day 4 , but the difference between the high

- dose and control groups was again not statistically significant

. In the absence of a statistically significant effect or dose

- related trend, the litter weight findings are not clearly related to treatment. No statistically significant differences among groups were observed for a ve rage offspring weight (measured on post

- partum days 1 and 4) and offspring body weight change per pup (between post

- partum days 1 and 4)

.

F 1 Offspring Necropsy: “ No treatment

- related macroscopic abnormalities were detected for 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. ” [McRae and Watson 2013, p. 41]

CONCLUSIONS

Systemic Toxicity: The L OAEL for systemic toxicity in males and females is 1980 mg test item/kg

- bw/day (872 mg a.i./kg

- bw/day), based on effects on hematology, clinical chemistry, and organ weights. In males, effects included reduced hemoglobin, erythrocyte count, hematocrit, and serum total protein, elevated albumin/globulin ratio, and decreased absolute and relative pituitary gland weights 1 . In females, effects included reduced serum calcium and inorganic phosphate, reduced total leukocyte and lymphocyte counts, increased eosinophil count, elevated absolute and relative

1 Although no treatment

- related histological changes were observed in the pituitary, the primary rationale for including decreased pituitary gland weight in high

- dose males as one of the determinant s of the LOAEL for systemic toxicity is the magnitude of the decrease (approximately 20.1% and 33.3% for absolute and relative pituitary weights, respectively , compared to controls ).Final HPV 2 Test Rule Data Adequacy Review (November 2013) CAS RN 31138

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14 thyroid/parathyroid weights 1 , and decreased absolute and relative combined uterus and cervix weights 2 . The NOAEL for systemic toxicity in males and females is 594 mg test item/kg

- bw/day (261 mg a.i./kg

- bw/day).

Reproductive Toxicity: The L OAEL for reproductive toxicity in males and females is 1980 mg test item/kg

- bw/day (872 mg a.i./kg

- bw/day) , based on reductions in litter size at birth

, decreased absolute and relative combined uterus and cervi x weights 3

, and decreased absolute and relative pituitary gland weight in males 4

. The NOAEL for reproductive toxicity in males and females is 594 mg test item/kg

bw/day (261 mg a.i./kg

- bw/day).

Developmental Toxicity: The L OAEL for developmental toxicity in males and females is 1980 mg test item/kg

- bw/day (872 mg a.i./kg

- bw/day) , based on reduced litter size at birth. The NOAEL for developmental toxicity in males and females is 594 mg test item/kg

- bw/day (261 mg a.i./kg

- bw/ day).

STUDY RELIABILITY

[1] Reliable without restrictions.

REFERENCES

McRae, L.A., Watson, P. 2013. Sodium Glucoheptonate (EC 250

- 480

- 2): Oral (Gavage) Combined Repeat Dose Toxicity Study w ith Reproduction/Developmental Toxicity Screening Test i n t he Rat

. Performing Laboratory: Harlan Laboratories Ltd., Derbyshire, UK. Project No. 41104692R. Sponsor: Harcros Chemicals Inc., Dalton, GA.

Text in quotes is taken verbatim from McRae and Watson ( 2013 )

.

1 Although no treatment

- related histological changes were observed in t he thyroid/parathyroid, the rationale for including increased thyroid/parathyroid weight in high

- dose females as one of the determinants of the LOAEL for systemic toxicity is the magnitude of the increase (approximately 44.7% and 40.0% for absolute and rel ative thyroid/parathyroid weights, respectively).

2 Although no treatment

- related histological changes were observed in the uterus/cervix, the decreased absolute and relative weight of the uterus/cervix seen in high

- dose females is included as one of the d eterminants of the LOAEL for systemic toxicity because “[d]ecreased or increased uterine weight” is an effect “on the uterus that may be considered adverse” ( http://www.epa.gov/raf/publications/guidelines

- reproductive

- tox

- risk

- assessment.htm p. 41).
3 Although no treatment

- related histological changes were observed in the uterus/cervix, the decreased absolute and relative weight of the uterus/cervix seen in h igh

- dose females is included as one of the determinants of the LOAEL for reproductive toxicity because uterine weight is considered a female

- specific endpoint of reproductive toxicity and “[d]ecreased or increased uterine weight” is an effect “on the uteru s that may be considered adverse” ( http://www.epa.gov/raf/publications/guidelines

- reproductive

- tox

- risk

- assessment.htm pp. 38, 41).

4 Although no treatment

- related histological changes were observed in the pituitary, rationales for including decreased pituitary gland weight in high

- dose males as a determinant of the LOAEL for reproductive toxicity are: (1) the magnitude of the decrease (approximately 20.1% and 33.3% for absolute and relative pituitary weights, respectively); and (2) pituitary weight is one of the male

- specific endpoint s of reproductive toxicity identified by EPA ( http://www.epa.gov/raf/publications/guidelines

- reproductive

- tox

- risk

- assessment.htm pp. 26

- 27).Final HPV 2 Test Rule Data Adequacy Review (November 2013) CAS RN 31138

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- 5

15 E

. DATA ADEQUACY SUMMARY TABLE

Endpoint Study Information Available Acceptable Data Gap

Aquatic Effects Acute Toxicity to Fish a Yes No Yes Acute Toxicity to Aquatic Invertebrates a Yes No Yes

Toxicity to Aquatic Plants a Yes Yes No Health Effects Acute Toxicity a Yes Yes No Repeated

- Dose Toxicity Yes Yes No Gene Mutation a Yes Yes No Chromosomal Aberrations a Yes Yes No Reproductive Toxicity Yes Yes No Developmental Toxicity Yes Yes No

a Data were reviewed in a previous report.