SRC TR-13-071

HPV2 TEST RULE DATA ADEQUACY REVIEW (FINAL):

REPEATED-DOSE, REPRODUCTIVE, AND DEVELOPMENTAL TOXICITY

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, 2013
Final HPV2 Test Rule Data Adequacy Review (November 2013) CASRN 31138-65-5

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TABLE OF CONTENTS

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

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

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

D. DATA FOR SIDS ENDPOINTS.............................................................................................. 3

D-1. Repeated-Dose, Reproductive, and Developmental Toxicity............................................ 3

E. DATA ADEQUACY SUMMARY TABLE........................................................................... 15
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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). 

The HPV2 test rule requires testing of CASRN 31138-65-5 for the following toxicological

endpoints: C1 (consisting of acute toxicity to fish, acute toxicity to Daphnia, and toxicity to 

algae testing if log Kow < 4.2 or chronic toxicity to Daphnia and toxicity to algae testing if log 

Kow ≥ 4.2), D (acute mammalian toxicity test), E1 (bacterial reverse mutation test), E2 

(chromosomal aberration or micronucleus test), and F1 (combined repeated-dose toxicity study 

with the reproduction/developmental toxicity screening test). Since the log Kow of CASRN 

31138-65-5 is estimated to be -6.441, 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 F1 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). 

These 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-dose, 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 included 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.
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C. SUMMARY OF DATA ADEQUACY FOR TESTING REQUIRED BY TEST RULE

Data supplied in the submission identified above are adequate to satisfy the combined repeateddose 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-HQOPPT-2007-0531-0715 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-OPPT2007-0531-0719 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 requirement on March 7, 2013 

(available online at http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT2007-0531-0709 as of November 1, 2013)

The data from the submissions above have been reviewed in separate documents (SRC Technical 

Reports #13-045 and #13-059). The data were determined to be adequate to satisfy the toxicity 

to plants (algae) (component of C1), acute mammalian toxicity (D), gene mutation (E1), and 

chromosomal aberrations (E2) testing requirements, but not adequate to satisfy the acute toxicity 

to fish (component of C1) or acute toxicity to Daphnia (component of C1) testing requirements. 

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 Harcros 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%).
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D. DATA FOR SIDS ENDPOINTS

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

Summary of repeated-dose toxicity endpoint: Adequate

Summary of reproductive toxicity endpoint: Adequate

Summary of developmental toxicity endpoint: Adequate

Study 1: Combined repeated-dose oral toxicity study with 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 with Reproduction/Developmental Toxicity Screening Test in the Rat

This 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% Polysaccharides 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/#!documentDetail;D=EPA-HQOPPT-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 31138-65-5
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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 Repeated-Dose Toxicity Study with the Reproduction/Developmental 

Toxicity Screening Test 

GLP compliant?: Yes

Year study performed: 2013

Test species: Wistar HanTM:RccHanTM:WIST strain rats from Harlan Laboratories UK Ltd., 

Oxon, UK; at initiation, rats were approximately 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], gestation, 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 [analyses were] 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.
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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, but the data showed non-homogeneity of means, the data were analyzed 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 these analyses, pairwise 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 using the 

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

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

data was applied, incorporating analysis of variance (ANOVA). If 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 analyzed incorporating the 

Kruskal-Wallis test which if significant, 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 nonparametric analyses.” [McRae and Watson 2013, p. 33] 

Test method/conditions remarks: 

Analyses of Test Item Formulations:

Based on stability 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 formulations was assessed visually. During the 

study, samples of the test item formulations (nominal concentrations of 0, 11.9, 118.8, and 396 

mg test item/mL, corresponding to doses of 0, 26.1, 261, 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. 
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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, 261, 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 males 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. Food 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). Following 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. 

Males were euthanized on day 43. Females were allowed 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 changes

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 

time 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 days 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. 

Food 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 pairing (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 

reactivity to auditory, visual, and proprioceptive stimuli. Measured sensory reactivity parameters 
Final HPV2 Test Rule Data Adequacy Review (November 2013) CASRN 31138-65-5

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included grasp response, vocalization, toe pinch, touch escape, pupil reflex, pinch reflex, tail 

pinch, and finger approach. 

All animals were evaluated in behavioral 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 leukocyte

count (i.e., white blood cell count, WBC), differential leukocyte counts (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 albumin, total protein, 

albumin/globulin ratio, total bilirubin, urea, creatinine, alkaline phosphatase, alanine 

aminotransferase, aspartate aminotransferase, glucose, total cholesterol, calcium, chloride, 

potassium, sodium, bile acids, and inorganic phosphorus.

Gross Necropsy and Histopathology:

All adults were subjected to a full external and internal examination and selected organs were 

weighed. Organ weights were recorded for 5 parental rats/sex/group for the adrenals, brain, 

spleen, heart, kidneys, liver, thymus, and thyroid with parathyroids. Additionally, organ weights 

were recorded for 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/pons), 

cecum, 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, prostate 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 bladder, 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.
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Reproductive/Developmental Toxicity:

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

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

observed completion of parturition. 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 days 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 reproductive parameters included pre-coital interval, mating 

index, pregnancy index, gestation length, parturition index, pre-implantation loss, postimplantation loss, live birth index, viability index, and sex ratio.

Clinical condition of the offspring was 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. Histological 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 numbers of uterine implantations and corpora lutea were recorded for 

each female upon examination of the uterus. 

RESULTS

Endpoint value(s): 

Systemic Toxicity:

LOAEL (male and female, systemic toxicity) = 1980 mg test item/kg-bw/day (= 872 mg a.i./kgbw/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. 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./kgbw/day).

Reproductive Toxicity:

LOAEL (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).
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Developmental Toxicity:

LOAEL (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 occurred 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 the 26.1 and 261 mg a.i./kg-bw/day dose groups and in one male in the 872 mg a.i./kgbw/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 posture. 

“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] had generalized scab formation from day 19 

onwards. Observations of this nature are commonly observed in group housed animals and 

result from social interaction and are therefore considered unrelated to test item toxicity.” 

[McRae and Watson 2013, p. 35]

Body Weight and Food/Water Consumption:

Group mean weight gains at week 2 were statistically 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 body weight gain in females treated with 872 mg a.i./kg-bw/day was slightly lower 

throughout the study compared with controls. However, the decrease 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 prepairing 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 relevance of the increase is not clear and the finding does not appear to be toxicologically 

relevant. 
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Hematology and Clinical Chemistry Measurements:

Statistically significant decreases in hemoglobin (p < 0.05), erythrocyte count (p < 0.05), and 

hematocrit (p < 0.01) were 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 10.7%, 9.1%, and 11.3% less than control means. 

Significant decreases in the mean total leukocyte 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 counts 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 females in the control and high-dose groups were 0.000 and 0.028 x 

109/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 suggestive 

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 significant 

reductions 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 males, 

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 individual 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 decreases in mean inorganic phosphate concentrations were 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 individual phosphate 

measurements at the mid-dose were within the historical control range. A significant decrease (p 

< 0.05) in mean calcium was also observed in females in the high-dose group; the decrease was

approximately 7.4% relative to the control group. A significant increase (p < 0.05) in mean 

plasma glucose was observed in females in the low-dose group, but the absence of a dose-related 

trend or similar effects on mean plasma glucose levels in other dose groups suggests that the 

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.
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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 moderate 

peribronchiolar intra-alveolar 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]

One female in the high-dose group had a mass under the right forelimb at necropsy, which was 

confirmed to be an adenocarcinoma. This finding may have been sporadic as no similar findings 

were seen in other animals of the high-dose group or other groups.

Mean absolute 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 increases (p < 0.05) in mean 

absolute and relative thyroid/parathyroid weights, with increases of approximately 44.7% and 

40.0% for absolute and relative thyroid/parathyroid weights, respectively, relative to controls. 

Statistically significant decreases (p < 0.05) in mean 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 approximately 14.3% and 15.3% lower, respectively, than the control group. 

No 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./kgbw/day) were reduced total protein, reduced creatinine (within historical control range), and 

increased albumin/globulin ratio in males and reduced inorganic phosphate 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 relatively 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 

protein 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 weights1. 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 the decrease (approximately 20.1% and 33.3% for absolute and relative 

pituitary weights, respectively, compared to controls). 
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effects at the high dose included reduced serum calcium and inorganic phosphate, reduced total 

leukocyte and lymphocyte counts, increased eosinophil count, elevated absolute and relative 

thyroid/parathyroid weights1, and decreased absolute and relative combined uterus and cervix 

weights2.

Reproductive/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./kgbw/day dose group gave birth to a live litter and successfully reared young to post-partum day 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 

number 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. Gestation 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 severe bilateral hypospermia and 

severe bilateral germinal epithelial atrophy found upon histopathological examination, which 

may at least partially explain the lack of pregnancy in this mid-dose female. All other females 

successfully mated and became pregnant.

Examination of Reproductive Organs: As stated above, statistically significant decreases (p < 

0.05) in mean absolute and relative combined uterus and cervix weights were observed in 

females in the high-dose group. Histopathology revealed no treatment-related changes in the 

uterus or cervix of the high-dose group or any other group. As indicated above, one male in the 

mid-dose group had small epididymides and small flaccid testes. Histopathological 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 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 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 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).
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epididymides and testes of any other treated males, these 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 clinical 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 this 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 21.8% 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 litters from the control group. Differences in

mean litter size were not statistically significant on post-partum days 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 average offspring weight (measured on post-partum 

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

F1 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 LOAEL 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 

weights1. 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 determinants 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). 
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14

thyroid/parathyroid weights1, and decreased absolute and relative combined uterus and cervix 

weights2. 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 LOAEL 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 cervix weights3, and decreased absolute and relative pituitary gland weight 

in males4. The NOAEL for reproductive toxicity in males and females is 594 mg test item/kgbw/day (261 mg a.i./kg-bw/day).

Developmental Toxicity:

The LOAEL 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 with Reproduction/Developmental Toxicity Screening 

Test in the 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 the 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 relative 

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 determinants 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 high-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 uterus 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 endpoints of reproductive toxicity identified by 

EPA (http://www.epa.gov/raf/publications/guidelines-reproductive-tox-risk-assessment.htm pp. 26-27).
Final HPV2 Test Rule Data Adequacy Review (November 2013) CASRN 31138-65-5

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E. DATA ADEQUACY SUMMARY TABLE

Endpoint Study Information 

Available

Acceptable Data Gap

Aquatic Effects

Acute Toxicity to Fisha Yes No Yes

Acute Toxicity to Aquatic 

Invertebratesa

Yes No Yes

Toxicity to Aquatic Plantsa Yes Yes No

Health Effects

Acute Toxicitya Yes Yes No

Repeated-Dose Toxicity Yes Yes No

Gene Mutationa Yes Yes No

Chromosomal Aberrationsa Yes Yes No

Reproductive Toxicity Yes Yes No

Developmental Toxicity Yes Yes No

a Data were reviewed in a previous report.