SRC TR-14-021

HPV3 TEST RULE DATA ADEQUACY REVIEW (FINALDRAFT):

REPEATED-DOSE TOXICITY

2-Oxiranemethanamine, N-[4-(2-oxiranylmethoxy)phenyl]-N-(2-oxiranylmethyl)-, 

CASRN 5026-74-4

Prepared by:

Amy Nguyen, Jennifer Rayner, William Richards, and Teresa Manyin

Defense and Environmental Solutions Division

SRC, Inc.

7502 Round Pond Road

North Syracuse, NY 13212

Contract No. EP-W-09-027

Task FG007.4.009.006

Submitted to:

OCSPP/OPPT/RAD (7403M)

U.S. Environmental Protection Agency

1200 Pennsylvania Avenue

Washington, D.C. 20460

Bryan Lobar, Project Officer

David Brooks, Work Assignment Manager

Amy Benson, Alternate Work Assignment Manager

July 22, 2014
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TABLE OF CONTENTS

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

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

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

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

D-1. Repeated-Dose Toxicity..................................................................................................... 4
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A. SOURCE MATERIAL USED FOR ASSESSMENT

This document evaluates a submission from Huntsman Corporation (dated June 23, 2014) that 

contains toxicity testing data submitted in response to the test rule, “Testing of Certain High 

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

referred to herein as HPV3 test rule; available online at http://www.regulations.gov/#!document

Detail;D=EPA-HQ-OPPT-2009-0112-0086 as of July 22, 2014), for 2-oxiranemethanamine, N-

[4-(2-oxiranylmethoxy)phenyl]-N-(2-oxiranylmethyl)- (CASRN 5026-74-4).

The HPV3 test rule requires the following toxicological testing for CASRN 5026-74-4: 

C2 (consisting of 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) and F1 (combined 

repeated-dose toxicity study with the reproduction/developmental toxicity screening test OR 

repeated-dose 28-day oral toxicity study and reproduction/developmental toxicity screening test). 

Since the log Kow of CASRN 5026-74-4 is estimated to be 0.871, which is < 4.2, the C2 aquatic 

toxicity testing requirement is considered to include acute toxicity to Daphnia and toxicity to 

algae testing.

On June 17, 2014, EPA received the following report for toxicological testing from Huntsman 

Corporation2, conducted with the proposed analog, m-(2,3-epoxypropoxy)-N-N-bis(2,3-

epoxypropyl)aniline (CASRN 71604-74-5) and submitted in response to the F1 testing 

requirement: 

 Beerens-Heijnen, C.G.M. 2013. Repeated dose 28-day oral toxicity study with m-(2,3-

epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline by daily gavage in the rat. WIL 

Research Europe B.V., The Netherlands, Project No. 501432. Sponsor: Huntsman 

Advanced Materials (Switzerland) GmbH, BASEL, Switzerland.

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 are evaluated for adequacy in Section D. The study was summarized 

according to a standard format and assigned a Klimisch reliability code based on its scientific 

merit and conduct, using professional judgment. To determine whether data were adequate to 

satisfy the test rule requirement 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. 

 

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 July 22, 2014.

2 Submitted in an email from Ms. Lynne Oreschnigg of Huntsman Corporation to Mr. John Schaeffer of EPA.
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C. SUMMARY OF DATA ADEQUACY FOR TESTING REQUIRED BY TEST RULE

Evaluation of Adequacy of Proposed Analog for Read-Across Strategy: 

In response to the F1 testing requirement for CASRN 5026-74-4, Huntsman submitted an oral 

repeated-dose study conducted with a proposed analog, CASRN 71604-74-5. The proposed 

analog is a positional isomer of CASRN 5026-74-4 and the two chemicals have identical 

functional groups. On the basis of these similarities, the proposed analog is reasonably expected 

to have toxicity similar to that of the test rule chemical. However, any further testing conducted 

to fulfill HPV3 testing requirements should be conducted with CASRN 5026-74-4, rather than 

CASRN 71604-74-5.

Evaluation of Toxicological Data Submission Dated June 17, 2014:

Data submitted for the 28-day repeated-dose toxicity study conducted with CASRN 71604-74-5, 

supplied by Huntsman and reviewed herein, are adequate to satisfy the repeated-dose toxicity 

component of the F1 testing requirement promulgated in the final HPV3 test rule. 

However, data submitted for the 28-day repeated-dose toxicity study conducted with CASRN 

71604-74-5, supplied by Huntsman and reviewed herein, are not adequate to satisfy the 

reproduction or developmental toxicity components of the F1 testing requirement promulgated in 

the final HPV3 test rule. 

Determination of Data Adequacy for the Reproductive/Developmental Toxicity (components of 

F1) Testing Requirement:

Based on the 28-day repeated-dose toxicity study data, Huntsman concluded in a previous 

submission (dated October 11, 2013, available online at http://www.regulations.gov/#!document

Detail;D=EPA-HQ-OPPT-2009-0112-0233 as of July 22, 2014) that a 

reproduction/developmental toxicity screening test (component of F1) for CASRN 5026-74-4 

was not needed, implying that the 28-day repeated-dose toxicity study of CASRN 71604-74-5 

would satisfy the F1 testing requirement for CASRN 5026-74-4. However, the submitted 

repeated-dose toxicity study conducted with CASRN 71604-74-5 is not adequate to satisfy the

reproduction/developmental toxicity component of the F1 testing requirement. As stated in the 

HPV3 test rule, the F1 testing requirement can be satisfied by conducting either (1) a combined 

repeated-dose toxicity study with the reproduction/developmental toxicity screening test or (2) a 

reproduction/developmental toxicity screening test and a repeated-dose 28-day oral toxicity 

study in rodents. Alternatively, EPA occasionally considers the combination of other specific 

tests acceptable to satisfy the reproduction/developmental toxicity component of F11. For 

example, the F1 testing requirement can be fulfilled with a 90-day repeated-dose toxicity study 

that sufficiently documents examination of the reproductive organs and finds no histological 

effects in male or female reproductive organs, in combination with an adequate developmental 

toxicity study. 

 

1 See EPA’s “Guidance for Meeting SIDS Requirements” (Section 5.7 “Toxicity to Reproduction”), available at 

http://www.epa.gov/chemrtk/pubs/general/sidsappb.htm as of July 22, 2014, which is based on the OECD Manual 

for the Assessment of Chemicals Chapter 4, Section 4.3 “Guidance for the Initial Assessment of Health Effects”, 

Sub-section 4.3.7 “Reproduction/Developmental Toxicity”, available at http://www.oecd.org/chemicalsafety/riskassessment/manualfortheassessmentofchemicals.htm as of July 22, 2014.
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The 28-day repeated-dose toxicity study conducted with CASRN 71604-74-5 found adverse 

effects on reproductive organs (including dose-related decreases in male and female reproductive 

organ weights, atrophy of female reproductive organs, and reduced contents of the seminal 

vesicles and coagulating glands), but did not assess potential consequences on reproductive 

function (e.g., mating, fertility, and parturition). Furthermore, the study did not address any 

developmental toxicity endpoints and thus is not considered adequate for assessing 

developmental toxicity. Therefore, a reproduction and developmental toxicity screening test 

(following 40 CFR 799.9355) using CASRN 5026-74-4 must be conducted to satisfy the 

reproduction and developmental toxicity components of the F1 testing requirement. 

Evaluation of Previously Submitted Toxicological Data:

In a previous submission (dated October 11, 2013; available online at http://www.regulations.

gov/#!documentDetail;D=EPA-HQ-OPPT-2009-0112-0233 as of July 22, 2014), Huntsman 

supplied data from chronic toxicity to Daphnia and toxicity to algae tests conducted with 

CASRN 5026-74-4. These data were reviewed in a separate report (SRC Technical Report #14-

009, available online at http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2009-

0112-0259 as of July 22, 2014) and were determined to be adequate to satisfy the acute toxicity 

to Daphnia1and toxicity to algae components of the C2 testing requirement.

Summary of Data Adequacy for Toxicological Testing Required by the Test Rule:

Data supplied by Huntsman for the chronic toxicity to Daphnia and toxicity to algae endpoints 

are considered adequate to satisfy the C2 testing requirement. Data supplied by Huntsman for 

the repeated-dose toxicity endpoint are considered adequate to satisfy the repeated-dose toxicity 

(component of F1) testing requirement. 

The reproductive and developmental toxicity endpoints (components of the F1 testing 

requirement) are considered data gaps. A reproduction and developmental toxicity 

screening test (following 40 CFR 799.9355) using CASRN 5026-74-4 must be conducted to 

satisfy the reproduction and developmental toxicity components of the F1 testing 

requirement. Prior to conducting the reproduction and developmental toxicity sceening 

test, a dose range-finding test must be conducted using CASRN 5026-74-4 to determine 

appropriate dose levels for the definitive test.

 

1 While acute toxicity to Daphnia (component of C2) data are not available, EPA considers the adequate chronic 

toxicity to Daphnia study supplied by Huntsman on October 11, 2013 acceptable to satisfy the acute toxicity to 

Daphnia component of the C2 testing requirement. 
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D. DATA FOR SIDS ENDPOINTS

D-1. Repeated-Dose Toxicity

Summary of endpoint: Adequate

Study 1: Repeated-dose oral toxicity study in rats (Beerens-Heijnen, 2013)

Title: Repeated dose 28-day oral toxicity study with m-(2,3-epoxypropoxy)-N,N-bis(2,3-

epoxypropyl)aniline by daily gavage in the rat.

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: m-(2,3-Epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline (CASRN 71604-74-5); 

supporting substance for 2-oxiranemethanamine, N-[4-(2-oxiranylmethoxy)phenyl]-N-(2-

oxiranylmethyl)- (CASRN 5026-74-4)

Remarks: Purity: 100%; Clear, slightly yellow, very viscous liquid; Batch No.: AAB0581300 

METHOD

Method/guideline followed: OECD TG 407 (2008) “Repeated-Dose 28-day Oral Toxicity 

Study in Rodents”; Commission Regulation (EC) No. 440/2008, B.7 “Repeated Dose (28 Days) 

Toxicity (Oral)”; OPPTS 870.3050 (2000) “Repeated Dose 28-day Oral Toxicity Study in 

Rodents”

Test type: Repeated-dose toxicity test

GLP compliant?: Yes

Year study performed: 2012

Test species: Wistar Crl:WI(Han) strain rats; obtained from Charles River Deutschland, 

Sulzfeld, Germany; age: ~6 weeks; weight at initiation: 151–183 g (males), 124–147 g (females) 

Number of animals/sex/dose: 5

Route of administration: Oral (gavage)

Vehicle: Propylene glycol

Exposure duration: 28 days
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Doses/concentrations tested: 0, 50, 150, and 450 mg/kg-bw/day1

Statistical methods used: 

“The following statistical methods were used to analyze the data:

 If the variables could be assumed to follow a normal distribution, the Dunnett-test 

(... many-to-one t-test) based on a pooled variance estimate was applied for the 

comparison of the treated groups and the control groups for each sex.

 The Steel-test (... many-to-one rank test) was applied if the data could not be assumed to 

follow a normal distribution.

 The Fisher Exact-test... was applied to frequency data.

 The Kruskal-Wallis nonparametric ANOVA test... was applied to motor activity data to 

determine intergroup differences. In case intergroup differences were seen, the Wilcoxon 

test... was applied to compare the treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of 

significance. Group means were calculated for continuous data and medians were calculated for 

discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact 

values for means and pooled variances.” [Beerens-Heijnen 2013, p. 15]

Test method/conditions remarks: 

Test Item Preparation and Analyses of Test Item Formulations: 

The test item was diluted in propylene glycol to prepare test concentrations within 6 hours prior 

to dosing each day. Formulations were stirred with a magnetic stirrer for at least 10 minutes

during dosing. Accuracy, stability, and homogeneity analyses of test item formulations in 

propylene glycol were conducted on day 19 of the study. Dose formulation concentrations, 

determined by HPLC with UV detection (wavelength of 253 nm), were 85–115% of nominal 

concentrations. Low- and high-dose test formulations were stable for at least 6 hours at room 

temperature. The low- and high-dose formulations were homogeneous as indicated by a ≤ 10% 

coefficient of variation of concentrations in samples taken from the mixing vessel at 90%, 50%, 

and 10% heights. 

Experimental Procedures:

Following a 5-day acclimatization period, groups of rats (5/sex/group) were orally administered 

the test item daily via gavage at dose levels of 0, 50, 150, or 450 mg/kg-bw/day in propylene 

glycol for 28 consecutive days. A control group was treated with the vehicle only. Animals 

were housed in groups of 5/sex except during locomotor activity monitoring, when animals were 

housed individually. Food and water were available ad libitum except during the locomotor 

activity measurements. Animals were euthanized on day 29. 

Clinical Observations, Body Weight, and Food/Water Consumption:

 

1 Doses were selected based on the results of a dose range-finding study in female rats with gavage exposure 

(summary provided in Appendix 5 of the study report). In this study, no effects were seen at 250 mg/kg-bw/day 

after 5 days of dosing. Lethargy, hunched posture, piloerection, salivation, reduced body weight gain, and reduced 

food consumption were seen at 500 mg/kg-bw/day after 10 days of dosing. Lethargy, hunched posture, piloerection, 

salivation, lean appearance, ptosis, body weight loss, reduced food consumption, and distended GI tract with gas and 

red foci in glandular mucosa of the stomach were seen at 1000 mg/kg-bw/day after 5 days of dosing. 
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Animals were examined at least twice daily for mortality and clinical signs of toxicity. Detailed 

clinical observations were performed cageside on all animals within 15 minutes after dosing and

weekly in a standard arena. Body weight and food consumption (g/animal/day) were recorded 

weekly throughout the study. Relative food consumption was calculated weekly as g/kg-bw/day. 

Food consumption data were collected per cage and statistical analyses were not conducted on 

food consumption measurements. Water consumption was not recorded.

Functional Observations:

All animals were observed for hearing ability, pupillary reflex, static righting reflex, grip 

strength, and locomotor activity during week 4 of treatment.

Hematology and Clinical Chemistry Analyses:

Blood samples for hematology and clinical chemistry evaluations were collected from all 

animals on the day of necropsy. Animals were fasted for up to 24 hours, but water was freely 

available. Hematological parameters evaluated included: white blood cell count, differential 

leukocyte count (neutrophils, lymphocytes, monocytes, eosinophils, and basophils, relative to 

white blood cells), red blood cell count, reticulocyte count (relative to red blood cells), red blood 

cell distribution width, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular 

hemoglobin, mean corpuscular hemoglobin concentration, platelets, prothrombin time, and 

activated partial thromboplastin time. The following clinical chemistry parameters were 

determined: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total 

protein, albumin, total bilirubin, urea, creatinine, glucose, cholesterol, sodium, potassium, 

chloride, calcium, inorganic phosphate, and bile acids. 

Gross Necropsy and Histopathology:

All animals were necropsied and examined for macroscopic abnormalities. Organ weights were 

determined for the adrenal glands, brain, epididymides, heart, kidneys, liver, ovaries, spleen, 

testes, thymus, uterus including cervix, prostate, seminal vesicles including coagulating glands, 

and thyroid including parathyroid. Relative organ weights were calculated as the ratio of

absolute organ weight to individual body weight at termination. The following tissues collected 

from the control and high-dose groups were examined microscopically: brain (cerebellum, midbrain, and cortex), cecum, colon, epididymides, eyes (including optic nerve if detectable and 

Harderian gland), heart, ileum, kidneys, lung, ovaries, rectum, sciatic nerve, skeletal muscle, 

spinal cord (cervical, midthoracic, and lumbar), sternum with bone marrow, thyroid including 

parathyroid (if detectable), trachea, urinary bladder, and all gross lesions. The following tissues 

were examined for all groups (males and/or females) based on findings at the high dose: thymus, 

stomach, duodenum, jejunum, Peyer’s patches, spleen, mesenteric lymph node, mandibular 

lymph node, adrenal glands, femur including joint, prostate gland, seminal vesicles, coagulating 

glands, liver, uterus, cervix, and vagina. The pancreatic lymph node was examined 

microscopically only in some groups and in a limited number of animals (see Tables 3 and 4).

Endpoint value(s):

LOAEL (systemic toxicity) = 50 mg/kg-bw/day, based on minimal hemorrhage of the duodenum 

in 4/5 males and 3/5 females, minimal to slight hypertrophy with villous stunting of the 

duodenum in all males and females, and increased neutrophil counts in females.

NOAEL (systemic toxicity) = Not established
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Results remarks:

Mortality and Clinical Observations: No mortality occurred during the study. All animals dosed 

with 450 mg/kg-bw/day displayed lethargy, hunched posture, piloerection, pale feces, lean 

appearance, and ptosis during the study period. At 150 mg/kg-bw/day, hunched posture, with or 

without lethargy, was seen in all animals on two treatment days. During the study, salivation 

was observed after dosing among all animals of the 50, 150, and 450 mg/kg-bw/day dose groups. 

Due to the lack of clarity about the timing of this finding relative to the time of gavage dose 

administration, its toxicological significance is unclear. Other clinical signs (rales, 

chromodacryorrhea, and scabs on the shoulder) were observed at a low incidence among most 

test substance-dosed groups and considered to be within the range of normal biological variation 

for rats of the age and strain.

Body Weight, Body Weight Gain, and Food Consumption: From week 2 onwards, weekly mean 

body weights of males were significantly (p < 0.01) decreased by 22-39% at 450 mg/kg-bw/day

relative to controls. Weekly mean body weight gains of dosed males, relative to controls, were

significantly (p < 0.05) decreased by 19-25% at 150 mg/kg-bw/day on days 8, 15, and 28 and 

were significantly (p < 0.01) decreased by 80-100% at 450 mg/kg-bw/day on days 8, 15, 22, and 

28. Reductions in weekly male body weights and body weight gains occurred in a dose-related 

manner. In females, weekly mean body weights were significantly (p < 0.01) decreased by 13-

21% at 450 mg/kg-bw/day from week 3 onwards. Female weekly mean body weight gains were

significantly (p < 0.01) decreased by 50-70% at 450 mg/kg-bw/day, relative to controls, on days 

8, 15, 22, and 28. Terminal body weights were significantly (p < 0.01) decreased in males and 

females at 450 mg/kg-bw/day, with reductions of 40% and 22%, respectively, relative to 

controls. 

In males, food consumption and relative food consumption were decreased by 48% and 21%, 

respectively, at 450 mg/kg-bw/day compared with controls. In females, food consumption and 

relative food consumption were decreased by 29% and 18%, respectively, at 450 mg/kg-bw/day

compared with controls. Statistical comparisons were not conducted for food consumption.

Functional Observations: Locomotor activity was significantly (p < 0.05) decreased in males and 

females at 450 mg/kg-bw/day only. In males, total movements were reduced by 60% relative to 

controls. In females, total movements and ambulations were reduced by 76% and 71%, 

respectively, relative to controls. No other effects on functional parameters were observed. 

Hematology: Hematological changes that reached statistical significance (p < 0.05) were 

observed in males at 150 and 450 mg/kg-bw/day and in females at 50, 150, and 450 mg/kgbw/day. The statistically significant hematological changes in males and females, and the 

calculated percent change in each parameter compared to controls, are provided below. 

Hematology changes for cell types marked with (#) were calculated by comparing cell type as % 

of total white blood cells in treated group with cell type as % of total white blood cells in control 

group. Hematology changes for cell types marked with (*) were calculated by comparing cell 

type as % of total red blood cells in treated group with cell type as % of total red blood cells in 

control group.
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Males:

Hematology changes at 150 mg/kg-bw/day included the following:

 Increased neutrophil count (#): 171%

 Decreased lymphocyte count (#): 23%

Hematology changes at 450 mg/kg-bw/day included the following:

 Increased neutrophil count (#): 580%

 Decreased lymphocyte count (#): 76%

 Decreased red blood cell count: 17%

 Increased reticulocyte count (*): 196%

 Increased red blood cell distribution width: 21%

 Decreased hemoglobin: 20%

 Decreased hematocrit: 17%

 Increased platelet count: 59%

Females:

Hematology changes at 50 mg/kg-bw/day included the following:

 Increased neutrophil count (#): 56%

Hematology changes at 150 mg/kg-bw/day included the following:

 Increased neutrophil count (#): 122%

Hematology changes at 450 mg/kg-bw/day included the following:

 Increased neutrophil count (#): 683%

 Increased monocyte count (#): 131%

 Decreased red blood cell count: 16%

 Increased reticulocyte count (*): 230%

 Decreased hemoglobin: 16%

 Decreased mean corpuscular hemoglobin concentration: 6%

 Increased platelet count: 39%

 Shortened activated partial thromoboplastin time: 21% 

Clinical Chemistry: Clinical chemistry changes that reached statistical significance (p < 0.05) 

were observed in males at 450 mg/kg-bw/day and in females at 150 and 450 mg/kg-bw/day. The 

statistically significant clinical chemistry changes in males and females, and the calculated 

percent change in each parameter compared to controls, are provided below.

Males:

Clinical chemistry changes at 450 mg/kg-bw/day included the following:

 Decreased albumin: 10%

 Increased total bilirubin: 69%

 Decreased creatinine: 12%

 Decreased glucose: 24%

 Increased cholesterol: 61%

 Increased bile acids: 170%
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 Decreased sodium: 1.3%

 Decreased chloride: 3%

Females: 

Clinical chemistry changes at 150 mg/kg-bw/day included the following:

 Increased total bilirubin: 46%

 Increased cholesterol: 81%

Clinical chemistry changes at 450 mg/kg-bw/day included the following:

 Increased total bilirubin: 42%

 Increased cholesterol: 132%

 Decreased albumin: 15%

 Decreased creatinine: 12%

 Decreased chloride: 3%

Organ Weights: Changes in absolute and relative organ weights were observed in males at 50, 

150, and 450 mg/kg-bw/day and in females at 150 and 450 mg/kg-bw/day. In males, the study 

author noted lower seminal vesicle weights at 50 mg/kg-bw/day, although the decreases of 19% 

(absolute) and 11% (relative) were not statistically significant. Dose-related decreases were also 

noted in absolute and relative thymus, ovary, and uterus weights in females starting at 50 mg/kgbw/day. The statistically significant (p < 0.05) organ weight changes in males and females, the 

calculated percent change in organ weights compared to controls, and the weight changes that 

were accompanied by corroborative adverse findings at macroscopic (*) or microscopic (#) 

examinations are listed below. 

Males: 

Organ weight changes at 50 mg/kg-bw/day included the following:

 Decreased absolute (33%) and decreased relative (25%, not significant) prostate weights

Organ weight changes at 150 mg/kg-bw/day included the following:

 Decreased absolute (42%) and decreased relative (36%) prostate weights

 Decreased absolute (31%) seminal vesicle weight 

 Increased relative (10%) liver weight 

 Increased relative (10%) kidney weight 

Organ weight changes at 450 mg/kg-bw/day included the following:

 Decreased absolute (13%) and increased relative (47%) brain weights

 Decreased absolute (33%) and increased relative (12%) heart weights

 Decreased absolute (85%) and decreased relative (75%) thymus weights*

 Increased relative (43%) liver weight 

 Increased relative (38%) thyroid weight 

 Decreased absolute (21%) and increased relative (33%) kidney weights*

 Decreased absolute (34%) and increased relative (130%) adrenal weights*#

 Decreased absolute (42%) spleen weight#

 Decreased absolute (14%) and increased relative (44%) testes weights
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 Decreased absolute (66%) and decreased relative (43%) prostate weights*

 Decreased absolute (21%) and increased relative (34%) epididymides weights

 Decreased absolute (73%) and decreased relative (56%) seminal vesicle weights*#

Females: 

Organ weight changes at 150 mg/kg-bw/day included the following:

 Decreased absolute (26%) and decreased relative (25%) thymus weights

Organ weight changes at 450 mg/kg-bw/day included the following:

 Increased relative (23%) brain weight 

 Increased absolute (22%) and increased relative (56%) liver weights#

 Decreased absolute (79%) and decreased relative (74%) thymus weights*#

 Increased relative (27%) kidney weight*

 Increased relative (66%) adrenal weight*#

 Decreased absolute (28%) spleen weight#

 Decreased absolute (54%) ovaries weight*

 Decreased absolute (77%) and decreased relative (70%) uterus weights*#

Gross Necropsy Findings: Selected incidences of macroscopic findings are summarized in Table 

1 (males) and Table 2 (females) by tissue and dose.

Table 1. Incidence of macroscopic findings in male rats*

Tissue Finding Dose (mg/kg-bw/day)

0 50 150 450

Emaciation 0/5 0/5 0/5 4/5

Forestomach

Irregular surface 0/5 0/5 5/5 4/5

Duodenum

Wall thickened/dilation 0/5 0/5 3/5 4/5

Gelatinous contents 0/5 0/5 3/5 4/5

Jejunum

Wall thickened/dilation 0/5 0/5 2/5 4/5

Gelatinous contents 0/5 0/5 2/5 4/5

Mesenteric Lymph Node

Discoloration (reddish/dark red/purple) 0/5 0/5 4/5 5/5

Pancreatic Lymph Node

Discoloration (reddish) 0/5 0/5 1/5 0/5

Enlargement 0/5 0/5 2/5 3/5

Adrenal Gland

Discoloration (pale) 0/5 0/5 0/5 4/5

Kidney

Discoloration (greenish) 0/5 0/5 0/5 5/5

Thymus

Reduced size 0/5 0/5 0/5 5/5

Prostate

Reduced size 0/5 0/5 0/5 5/5

Seminal Vesicle

Reduced size 0/5 0/5 0/5 5/5
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* Data from Appendix 4 “Histopathology Report”

Table 2. Incidence of macroscopic findings in female rats*

Tissue Finding Dose (mg/kg-bw/day)

0 50 150 450

Emaciation 0/5 0/5 0/5 5/5

Forestomach

Irregular surface 0/5 0/5 3/5 5/5

Focus/foci (yellowish/tan, red/dark red) 0/5 0/5 0/5 3/5

Duodenum

Wall thickened/dilation 0/5 0/5 3/5 4/5

Gelatinous contents 0/5 0/5 2/5 4/5

Jejunum

Wall thickened/dilation 0/5 0/5 2/5 4/5

Gelatinous contents 0/5 0/5 1/5 5/5

Mesenteric Lymph Node

Discoloration (reddish/dark red/purple) 0/5 0/5 2/5 4/5

Pancreatic Lymph Node

Discoloration (reddish) 0/5 1/5 2/5 0/5

Enlargement 0/5 0/5 1/5 4/5

Adrenal Gland

Discoloration (pale) 0/5 0/5 1/5 4/5

Kidney

Discoloration (greenish) 0/5 0/5 0/5 5/5

Thymus

Reduced size 0/5 0/5 0/5 4/5

Uterus

Reduced size 0/5 0/5 1/5 5/5

Ovary

Reduced size 0/5 0/5 0/5 3/5

* Data from Appendix 4 “Histopathology Report”

Histopathology: Histological changes were observed at ≥ 50 mg/kg-bw/day. Selected incidences

of histological findings are summarized in Table 3 (males) and Table 4 (females) by tissue, 

grade, and dose. 

Table 3. Incidence of histological findings in male rats*

Tissue Finding Grade Dose (mg/kg-bw/day)

0 50 150 450

Stomach

Ulcer, forestomach Total 0/5 0/5 2/5 5/5

slight 0/5 0/5 1/5 0/5

moderate 0/5 0/5 1/5 1/5

marked 0/5 0/5 0/5 4/5

Hyperplasia, forestomach Total 0/5 1/5 5/5 5/5

minimal 0/5 1/5 0/5 0/5

slight 0/5 0/5 3/5 2/5

moderate 0/5 0/5 2/5 3/5

Duodenum

Hemorrhage Total 0/5 4/5 3/5 5/5
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minimal 0/5 4/5 3/5 2/5

slight 0/5 0/5 0/5 3/5

Hypertrophy with villous stunting Total 0/5 5/5 5/5 5/5

minimal 0/5 4/5 0/5 0/5

slight 0/5 1/5 5/5 1/5

moderate 0/5 0/5 0/5 4/5

Jejunum

Hemorrhage Total 0/5 0/5 0/5 3/5

minimal 0/5 0/5 0/5 2/5

slight 0/5 0/5 0/5 1/5

Hypertrophy with villous stunting Total 0/5 0/5 2/5 5/5

minimal 0/5 0/5 1/5 0/5

slight 0/5 0/5 1/5 3/5

moderate 0/5 0/5 0/5 2/5

Peyer’s Patches

Lymphoid atrophy Total 0/5 0/5 1/4 3/3

minimal 0/5 0/5 0/4 2/3

slight 0/5 0/5 1/4 1/3

Mesenteric Lymph Node

Lymphoid atrophy Total 0/5 0/5 3/5 5/5

minimal 0/5 0/5 3/5 1/5

slight 0/5 0/5 0/5 2/5

moderate 0/5 0/5 0/5 2/5

Increased mast cells Total 0/5 0/5 5/5 1/5

slight 0/5 0/5 3/5 1/5

moderate 0/5 0/5 2/5 0/5

Erythrophagocytosis Total 0/5 1/5 5/5 5/5

slight 0/5 1/5 3/5 0/5

moderate 0/5 0/5 2/5 5/5

Thymus

Lymphoid atrophy Total 0/5 0/5 0/5 5/5

minimal 0/5 0/5 0/5 1/5

slight 0/5 0/5 0/5 3/5

marked 0/5 0/5 0/5 1/5

Spleen

Hemopoietic foci Total 5/5 2/5 5/5 5/5

minimal 5/5 2/5 5/5 2/5

slight 0/5 0/5 0/5 3/5

Lymphoid atrophy Total 0/5 0/5 0/5 5/5

minimal 0/5 0/5 0/5 1/5

slight 0/5 0/5 0/5 4/5

Adrenal Glands

Vacuolization, zona fasciculata Total 0/5 1/5 1/5 4/5

minimal 0/5 1/5 1/5 2/5

slight 0/5 0/5 0/5 2/5

Pancreatic Lymph Node

Lymphoid hyperplasia Total 0/0a 0/0a 2/2 3/3

minimal 0/0a 0/0a 1/2 0/3

slight 0/0a 0/0a 0/2 1/3

moderate 0/0a 0/0a 1/2 1/3

marked 0/0a 0/0a 0/2 1/3

Mandibular Lymph Node
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Lymphoid atrophy Total 0/5 0/5 1/5 4/4

Seminal vesicles

Reduced contents Total 1/5 0/5 0/5 5/5

slight 0/5 0/5 0/5 5/5

moderate 1/5 0/5 0/5 0/5

Coagulating glands

Reduced contents Total 0/5 0/5 0/5 5/5

slight 0/5 0/5 0/5 5/5

Femur

Decreased bone Total 0/5 0/5 0/5 5/5

slight 0/5 0/5 0/5 2/5

moderate 0/5 0/5 0/5 3/5

* Data from Appendix 4 “Histopathology Report”

a No pancreatic lymph nodes from this group were examined microscopically.

Table 4. Incidence of histological findings in female rats*

Tissue Finding Grade Dose (mg/kg-bw/day)

0 50 150 450

Stomach

Ulcer, forestomach Total 0/5 0/5 0/5 5/5

marked 0/5 0/5 0/5 5/5

Hyperplasia, forestomach Total 0/5 0/5 5/5 5/5

minimal 0/5 0/5 3/5 0/5

slight 0/5 0/5 1/5 0/5

moderate 0/5 0/5 1/5 4/5

marked 0/5 0/5 0/5 1/5

Duodenum

Hemorrhage Total 0/5 3/5 4/5 4/5

minimal 0/5 3/5 1/5 2/5

slight 0/5 0/5 3/5 2/5

Hypertrophy with villous stunting Total 0/5 5/5 5/5 5/5

minimal 0/5 2/5 1/5 0/5

slight 0/5 3/5 3/5 4/5

moderate 0/5 0/5 1/5 1/5

Jejunum

Hemorrhage Total 0/5 0/5 0/5 3/5

slight 0/5 0/5 0/5 3/5

Hypertrophy with villous stunting Total 0/5 0/5 1/5 5/5

minimal 0/5 0/5 1/5 1/5

slight 0/5 0/5 0/5 4/5

Peyer’s Patches

Lymphoid atrophy Total 0/5 1/5 1/5 4/5

minimal 0/5 0/5 0/5 1/5

slight 0/5 1/5 0/5 3/5

moderate 0/5 0/5 1/5 0/5

Mesenteric Lymph Node

Lymphoid atrophy Total 0/5 0/5 2/5 4/5

minimal 0/5 0/5 2/5 2/5

slight 0/5 0/5 0/5 2/5

Increased mast cells Total 0/5 0/5 3/5 2/5

slight 0/5 0/5 3/5 2/5
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Erythrophagocytosis Total 0/5 1/5 3/5 5/5

minimal 0/5 1/5 0/5 0/5

slight 0/5 0/5 3/5 3/5

moderate 0/5 0/5 0/5 2/5

Thymus

Lymphoid atrophy Total 0/5 0/5 0/5 5/5

minimal 0/5 0/5 0/5 1/5

slight 0/5 0/5 0/5 2/5

moderate 0/5 0/5 0/5 2/5

Liver

Hypertrophy, centrilobular Total 0/5 0/5 1/5 5/5

minimal 0/5 0/5 1/5 1/5

slight 0/5 0/5 0/5 4/5

Spleen

Hemopoietic foci Total 1/5 2/5 2/5 5/5

minimal 1/5 2/5 2/5 4/5

slight 0/5 0/5 0/5 1/5

Adrenal Glands

Vacuolization, zona fasciculata Total 0/5 0/5 0/5 4/5

minimal 0/5 0/5 0/5 1/5

slight 0/5 0/5 0/5 3/5

Pancreatic Lymph Node

Erythrophagocytosis Total 0/0a 1/1 2/3 0/4

slight 0/0a 1/1 1/3 0/4

moderate 0/0a 0/1 1/3 0/4

Lymphoid hyperplasia Total 0/0a 0/1 3/3 4/4

slight 0/0a 0/1 2/3 1/4

moderate 0/0a 0/1 1/3 3/4

Mandibular Lymph Node

Lymphoid atrophy Total 0/5 0/5 0/5 5/5

Uterus

Atrophy Total 0/5 0/5 2/5 5/5

Cervix

Atrophy Total 0/5 0/5 2/5 5/5

Vagina

Atrophy Total 0/5 0/5 2/5 5/5

Femur

Decreased bone Total 0/5 0/5 0/5 5/5

minimal 0/5 0/5 0/5 1/5

slight 0/5 0/5 0/5 3/5

moderate 0/5 0/5 0/5 1/5

* Data from Appendix 4 “Histopathology Report”

a No pancreatic lymph nodes from this group were examined microscopically.

The study author concluded that the LOAEL for local effects was 50 mg/kg-bw/day, the lowest 

dose tested, “based on the local effects in the gastrointestinal tract (duodenum, Peyer’s patches 

and lymph nodes), likely to be secondary effects due to the irritation potential of the test 

substance”. [Beerens-Heijnen 2013, p. 22] The study author concluded that the LOAEL for 

systemic toxicity was 150 mg/kg-bw/day, based on atrophy of the female reproductive organs 

(uterus, cervix, and vagina). The reviewer disagrees with the categorization of the histological 

changes in the gastrointestinal tract as merely local and/or evidence of irritation. The reviewer 
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considers the LOAEL for systemic toxicity to be 50 mg/kg-bw/day. A basis for this conclusion 

is provided below. 

Rationale for Determination of the LOAEL Value for Systemic Toxicity

(1) Microscopic examination of the duodenum showed hemorrhage in 4/5 males and 3/5 females

at 50 mg/kg-bw/day and hypertrophy with villous stunting in all males and females at 50 mg/kgbw/day. In contrast, the jejunum showed no adverse histologic changes at this dose and the 

forestomach showed hyperplasia in one male but no histological findings in females at the low 

dose (also, no treatment-related histological changes were seen in the ileum, which was 

examined microscopically only at 0 and 450 mg/kg-bw/day). The differences in expression of 

adverse histological effects in distinct sections of the gastrointestinal tract support the 

designation of the duodenum as a target organ. Internal bleeding/hemorrhage of the duodenum 

at the low dose, with higher severities at the mid and high doses, is considered a toxicologically 

relevant systemic effect. Shortening of the microvilli of the duodenum (villous stunting) at the 

low dose, with higher severities at the mid and high doses, is also considered a toxicologically 

relevant systemic effect because villous stunting is expected to result in decreased absorption of 

nutrients from the small intestine. The decrease in absorption of nutrients from the small 

intestine due to shortening of the microvilli is expected to adversely affect animal health. 

(2) By histologic examination, lymphoid atrophy in tissues of the immune system [Peyer’s 

patches (found in wall of the small intestine), spleen, thymus, and mesenteric and mandibular 

lymph nodes] was clearly evident only at the mid and high doses. Lymphoid atrophy results 

from necrosis or apoptosis of cells in lymphatic tissues

1

. Since nutrient deprivation and 

malnutrition generally lead to atrophy of lymphoid tissues including thymus, spleen, and lymph 

nodes,2,3the lymphoid atrophy observed in the current study may have resulted from the 

inhibition of nutrient uptake from the small intestine due to villous stunting in the duodenum and 

jejunum. As such, the microscopic changes observed in the small intestine appear to lead to 

atrophy of the lymphoid tissues at the mid and high doses. Therefore, the changes in the 

duodenum observed at the low dose may be indicative of the initiation of a progressive cascade 

of adverse effects in response to the test substance.

(3) Increases in neutrophil counts at ≥ 150 mg/kg-bw/day in males and ≥ 50 mg/kg-bw/day in 

females support a conclusion that inflammation is one component of the adverse effects induced 

by the test substance. The relationship of the inflammatory response to the histological changes 

in the duodenum and/or lymphoid tissues is unclear; however, the dose-response relationship and 

magnitude of the changes in neutrophil counts support the inclusion of this change in the basis 

for the LOAEL. 

 

1 Elmore, S.A. 2006. Histopathology of the lymph nodes. Toxicol. Pathol. 34: 425-454.

2 Beisel, W.R. 1996. Nutrition and immune function: Overview. J. Nutrition 126: 2611S-2615S.

3 Howard, J.K., Lord, G.M., Matarese, G., Vendetti, S., Ghatei, M.A., Ritter, M.A., Lechler, R.I., Bloom, S.R. 1999. 

Leptin protects mice from starvation-induced lymphoid atrophy and increases thymic cellularity in ob/ob mice. J. 

Clin. Invest. 104: 1051-1059.
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CONCLUSIONS

In this 28-day repeated-dose oral toxicity study, the LOAEL for systemic toxicity in both sexes 

was 50 mg/kg-bw/day, the lowest dose tested, based on minimal hemorrhage of the duodenum in 

4/5 males and 3/5 females, minimal to slight hypertrophy with villous stunting of the duodenum 

in all males and females, and increased neutrophil counts in females. A NOAEL for systemic 

toxicity was not established.

STUDY RELIABILITY

[2] Reliable with restrictions. Terminal body weights of three females in the low-dose group 

were not determined prior to necropsy. Some values listed for the macroscopic findings for 

males and females in Appendix 1 do not match the macroscopic findings found in Appendix 4. 

Within Appendix 4, the descriptions of the microscopic lesions are not presented consistently 

(for example, the lesion hypertrophy with villous stunting in the duodenum).

REFERENCES

Beerens-Heijnen, C.G.M. 2013. Repeated dose 28-day oral toxicity study with m-(2,3-

epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline by daily gavage in the rat. WIL Research 

Europe B.V., The Netherlands, Project No. 501432. Sponsor: Huntsman Advanced Materials 

(Switzerland) GmbH, BASEL, Switzerland. Study report not available online as of July 22, 

2014.