### SRC TR

- 1 4

- 021

HPV3 TEST RULE DATA ADEQUACY REVIEW ( FINAL DRAFT ):

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

Ju ly 22 , 2014Final

Draft HPV

3 Test Rule Data Adequacy Review ( July 2014) CAS RN 5026

- 74

- 4

i 

### TABLE OF CONTENTS

A. SOURCE MATERIAL USED FOR ASSESSMENT

................................ .............................. 1

B. EVALUATING DATA ADEQUACY

................................ ................................ ..................... 1

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

........ 2

D. DATA FOR SIDS ENDPOINTS

................................ ................................ .............................. 4

D

- 1. Repeated

- Dose Toxicity

................................ ................................ ................................ ..... 4Final

Draft HPV

3 Test Rule Data Adequacy Review ( July 2014) CAS RN 5026

- 74

- 4

1 

### 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 Ju ly 22 , 2014)

, for 2

- oxiranemeth anamine, 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 K

ow 

### < 4.2 or

chronic toxicity to Daphnia and toxicity to algae testing if log K

ow 

### ≥ 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/devel opmental toxicity screening test).

Since the log K

ow 

### of CASRN 5026

- 74

- 4 is estimated to be 0.87 1 

### , 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, 20 14, EPA received the following report for toxicological test ing from Huntsman

Corporation 2



### , 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 e valuated for adequacy in S ection 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 requiremen t 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 htt p://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.Final

Draft HPV

3 Test Rule Data Adequacy Review ( July 2014) CAS RN 5026

- 74

- 4

2 

### C. SUMMARY OF DATA ADEQUACY FOR TESTING REQUIRED BY TEST RUL E

Evaluation of Adequacy of Proposed Analog for Read

- Across Strategy:

In response to the F1 testing requirement for CASRN 5026

- 74

- 4, Huntsman submitte d 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 simil ar to that of the test rule chemical. However, a ny 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 Submi ssion Dated June 17 , 201 4 :

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 t est rule.

However, d ata 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 component s 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 c oncluded 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 Ju ly 2 2 , 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

- 7 4

- 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 requireme nt 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 r odents. Alternatively, EPA occasionally considers the combination of other specific

tests acceptable to satisfy the reproduction/developmental toxicity component of F1 1 

### . 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 a n 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/risk

assessment/manualfortheassessmentofchemicals.htm as of July 22, 2014.Final

Draft HPV

3 Test Rule Data Adequacy Review ( July 2014) CAS RN 5026

- 74

- 4

3 

### 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 content s 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 c onsidered adequate for assessing

developmental toxicity. Therefore, a reproducti on 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 compone nts 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.regul ations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2009

-

0112

- 0259 as of July 2 2 , 2014 ) and were determined to be adequate to satisfy the acute toxicity

to Daphnia 1 

### and 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 t oxicity 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 reproductio n 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.Final

Draft HPV

3 Test Rule Data Adequacy Review ( July 2014) CAS RN 5026

- 74

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4 

### 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

.

Th is study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: m

- (2,3

- E poxypropoxy)

- N,N

- bis(2,3

- epoxypropyl)aniline (CASRN 71604

- 74

- 5) ;

s upporting 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 4 07 ( 2008 ) “ Repeated

- Dose 28

- day Oral Toxicity

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

Toxicity (O ral)”; OPPTS 870.3050 (2000) “Repeated Dose 28

- day Oral Toxicity Study in

Rodents”

Test type: Repeated

- d ose t oxicity t est

GLP compliant?: Yes

Year study performed: 201 2

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)

, 1 24

– 147 g (females)

Number of animals/sex/dose: 5

Route of administration: Oral ( gavage )

Vehicle: Propylene glycol

Exposure duration: 28 daysFinal

Draft HPV

3 Test Rule Data Adequacy Review ( July 2014) CAS RN 5026

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5 

### Doses/concentrations tested: 0, 5 0, 150 , and 450 mg/kg

- bw/day 1



### 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 group s 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 a ctivity 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 l evel 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

- Hei jnen 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

. F ormulations 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 2 53 nm)

, were 85

– 115% of nominal

concentrations

. Low

- and high

- dose test formulation s were stable for at least 6 hours at room

temperature

. Th e 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, g roups of r ats ( 5 /sex/group) were orally administered

the test item daily via g avage 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 e xcept 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 sele cted 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, p iloerection, 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.Final

Draft HPV

3 Test Rule Data Adequacy Review ( July 2014) CAS RN 5026

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6 

### A nimals 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 d ata 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 faste d 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, potassi um,

chloride, calcium, inorganic phosphate, and bile acids

.

Gross Necropsy and Histopathology:

All animals were necropsied and examined for macroscopic abnormalities. O rgan 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 weight s were calculated as the ratio of

absolute organ weight to individual body weight at termination

. The following tissues collected

from th e control and high

- dose groups were examined microscopically: brain (cerebellum, mid

brain, and cortex), cecum, colon, epididymides, eyes (including optic nerve if detectable an d

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 hy pertrophy with villous stunting of the

duodenum in all males and females

, and increased neutrophil counts in females.

NOAEL (systemic toxicity) = Not establishedFinal

Draft HPV

3 Test Rule Data Adequacy Review ( July 2014) CAS RN 5026

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7 

### Results remarks:

Mortality and Clinical Observations : No mortalit y 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, h unched posture, with or

without lethargy, was seen in all animals on two treatment days. During th e study, s alivation

was observed after dosing among all animals of the 50, 150, and 450 mg/kg

- bw /day dose group s

. 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 in cidence among most

test substance

- dose d 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 weight s of males w ere significantly (p < 0.01) decreased by 22

- 39% at 450 mg/kg

- bw/day

relative to controls. W eekly mean body weight gain s of dosed males , relative to controls, w ere

significantly (p < 0.0 5 ) decreased by 19

- 25% at 150 mg/kg

- bw/day on days 8, 15, and 28 and

were significantly (p < 0.01) decreased by 8 0

- 100% at 450 mg/kg

- bw/day on days 8, 15, 22, and

28

. Reductions in weekly male body weight s and body weight gain s occurred in a dose

- related

manner. In females, weekly mean body weight s w ere significantly (p < 0.01) decreased by 13

- 21% at 450 mg/kg

- bw/day from week 3 onwards. Female weekly mean body weight gain s w ere

significantly (p < 0.01) decreased by 50

- 70% at 450 mg/kg

- bw/day , relative to controls , on days

8, 15, 22, and 28

. T erminal 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, fo od 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

. S tatistical comparisons were not condu cted for food consumption.

Functional Observations : Locom otor 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/kg

bw/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 cell s in treated group with cell type as % of total white blood cell s 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.Final

Draft HPV

3 Test Rule Data Adequacy Review ( July 2014) CAS RN 5026

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8 

### 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 (#) : 68 3 %

 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%

 S hortened activated partial thromoboplastin time: 21%

Clinical C hemistry : 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 cal culated

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%Final

Draft HPV

3 Test Rule Data Adequacy Review ( July 2014) CAS RN 5026

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9 

###  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 a bsolute and relative organ weight s 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, t he study

author noted lower seminal vesicle weight s at 50 mg/kg

- bw/day, although the decrease s of 19%

(absolute) and 11% (relative) w e re not statistically significant. Dose

- re lated decreases were also

noted in absolute and relative thymus, ovary, and uterus weights in females starting at 50 mg/kg

bw/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 finding s at macroscopic (*) or microscopic (#)

examinations are listed bel ow.

Males:

Organ weight changes at 50 mg/kg

- bw/day included the following:

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

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 (3 8%) 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 weightsFinal

Draft HPV

3 Test Rule Data Adequacy Review ( July 2014) CAS RN 5026

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10 

###  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:

Or gan 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 i ncidences of m acroscopic findings are summarized in Table

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

Table 1. Incidence of macroscopic findings in male rat s *

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

G elatinous 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 r ed/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 5/5Final

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### * Data from Appendix 4 “ Histopathology Report ”



### Table 2. Incidence of macroscopic findings in female rat s *

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

F ocus/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 (re d dish) 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 i ncidence s

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

grade, and dose.

Table 3

. I ncidence of h istological findings in male rat s *

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 5/5Final

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12 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

Hypertroph y 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 Tota l 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 Tota l 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 0/0 a 0/0 a 2/ 2 3/ 3

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

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

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

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

Mandibular Lymph NodeFinal

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13 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 rat s *

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 2/5Final

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14 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 0/0 a 1/ 1 2/ 3 0/ 4

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

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

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

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

moderate 0/0 a 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 irrita tion 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 disag rees with the categorization of the histological

changes in the gastrointestinal tract as merely local and/or evidence of irritation. The reviewerFinal

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15 

### considers the LOAEL for systemic toxicity to be 50 mg/kg

- bw/day. A basis for this conclusion

is provided be low.

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/kg

bw/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 gastrointestin al 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 th e 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

nutri ents 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 tissue s 1 

### . Since nutrient deprivation and

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

nodes, 2

, 3 

### the lymphoid atrophy observed in the current study may have resulted from the

inhibition of nutrient uptake from th e 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 ob served 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 conclusi on 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 relationsh ip 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. Nut rition 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.Final

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16 

### 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 l owest dose tested, based on minimal hemorrhage of the duodenu m in

4/5 males and 3/5 females

, minimal to slight hypertrophy with villous stunting of the duo denum

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, th e lesion hypertrophy with villous stunting in the duodenum).

REFERENCE S

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 Ju ly 22

, 2014.