Key. X Not Remarkable N= No Section I= lncomplete A= Autolysis t= minimal P= mild 3: modeiate 4aevere P= Present B= Benign M= Malignant m= missing one paired organ u= unscheduled sac. ldeath 549of619 HISTOPATHOLOGY INCIDENCE TABLE GROUP WIL 417002 90 Day Sacrifice Male Rat A N M A L Duct Inflammatory Cell Exudate, Medial Meatus Mineral Deposition VacuolizatiorXyst. Olfactory Epithelium i

6 9 3 0 4 h

z

I       2 1 1 1 1 * 1 1 1 1 _ 2 2 1 EPLl Experimental Pathology Laboratories, Inc.

6 9 3 1 3

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I

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1 1 _ 1 1 2 2 1 1 I 2 2 1

6 9 3 4 6

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t II 18 Key: X= Not Remarkable N= No Section I Incomplete A= Autolysts l= minimal2= mdd B= moderate 4= severe P= Present B= Semgn M= Malignant m= mlssing one paired organ u= unscheduled sac/ death 550 of 619 HISTOPATHOLOGY INCIDENCE TABLE GROUP WIL 417002 90 Day Sacrifice Male Rat A N I M 6 A 9 L 3 0 A OPTIC NERVE Degeneration, Vacuolar PITUITARY Hypertrophy, Pars Distalis PROSTATE X Atrophy, Acinar Epithelium Hyperplasia, Acinar Epithelium Infiltrate, Mononuclear Cell I SALIVARY GLAND, MANDIBULAR ) X I SEMINAL VESICLE Reduced Secretion X SKELETAL MUSCLE (RECTUS FEMORIS) X Degeneration I SKIN IX Inflammation, Preputial Gland h I SPINAL CORD, CERVICAL IX I EPL( 7

6 9 3 0 1.

1 x 6 6 9 9 3 3 1 2 Jai r II 19 XX XX XX li_ X XX XX X 1 XX li_ X

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x E

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x h x h

x 4

x h x E

s zc

s x

x x

I / I I 1 I /

I I , I / I I I I I I Experimental Pathology Laboratories, Inc. Key: X= Not Remarkable N= No Section I Incomplete A Autolysis t= minimal P= mild 3= moderate 4= s.?~ ere P Present E= Benign M= Malignant m= m& ng one paired organ u= unscheduled sac./ death 551 of 619 HISTOPATHOLOGY INCIDENCE TABLE GROUP  T : 3 2 7 K

x h

f E

h E

c

z

n

I

t   WIL 417002 90 Day Sacrifice Male Rat I SPINAL CORD, THORACIC IX I SPLEEN Accessory Tissue X STOMACH Edema, Nonglandular Hyperplasia, Squamous Epithelium, Nonglandular Inflammation, Nonglandular X I TESTIS IX I THYMUS Congestion 3 Necrosis, Cortical Lymphocytes THYROID X TRACHEA X Infiltrate. Mononuclear Cell Inflammation, Granulomatous Mineral Deposition URINARY BLADDER Infiltrate, Mononuclear Cell x I

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n n

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x n

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II 20 Key: X= Not Remarkable N= No Section l= lncomplete A= Autolysis l= mmimal P= mild 3= moderate 4= severe P= Present B= Benign M= Malignant m= missing one paired organ u= unscheduled sac/ death 552 of 619 HISTOPATHOLOGY INCIDENCE TABLE WIL 417002 90 Day Sacrifice Female Rat A N 6 9 3 I BONE MARROW, STERNUM IX I I BONE, STERNUM IX I .BRAlN X ESOPHAGUS X eve "

1 intraocular Retinal Atrophy Retinal Dysplasia INTESTINE LARGE, COLON X Nematode Parasite, Lumen EPLl II 21

6 9 3 6 6 n

n

x x x E

T x 2.

x

z x

n     GROUP 1 Experimental Pathology Laboratories, Inc. Key: X= Not Remarkable N= No Section I= lncompleie A= Autolysis l= minimal Z= mild 3= moderate 4= severe P= Present B= Benign M= Malignant m= missing one paired organ u= unschedukd %x./ death 553 of 619 HISTOPATHOLOGY INCIDENCE TABLE GROUP 1 WIL 417002 90 Day Sacrifice Female Rat II 22

Experimental Pathology Laboratories, Inc. Key: X= Not Remarkable N= No Section I= lncompkte A= Autolysis l= minimal2= mild S= moderate 4= severe P= Present B= Benign M= Malignant m= missing one paired organ u= unscheduled %x./ death 554 of 619 HISTOPATHOLOGY INCIDENCE TABLE GROUP

6 : 5 4    r  x      "  c  L WIL 417002 go Day Sacrifice Female Rat A N ti A L 1 1 z x x 4 x x $I Y x LYMPH NODE, MANDIBULAR Congestion/ Hemorrhage LYMPH NODE, MEDIASTINAL Conqestionn lemorrhaqe LYMPH NODE, TRACHEOBRONCHIAL Congestion/ Hemorrhage MAMMARY GLAND :

:

EPL] II 23 Experimental Pathology Laboratories, Inc. Key: X= Not Remarkable N= No Seciion I= lncompkte A= AutoIvsis l= minimal P mild 3= moderate d= severe P= Present B= Ben! gn M= Malignant m= misSing one pared organ u= unscheduled sac. ldeath 555 of 619 HISTOPATHOLOGY INCIDENCE TABLE WIL 417002 90 Day Sacrifice Female Rat A N A 9 L 3 5 / Medial Meatus / 1 / Mineral Deposition T GROUP 1

6 9 4 1 6

n

I

I

6 9 4 1 s

I? I

1

1

I EPLl II 24 Experimental Pathology Laboratories, Inc. Key: X= Not Remarkable N No Section I= lncamplete A= Autolysis 1 =minmai l= mild 3= nwderate 4= severe P= Present B= Benign M= Malignani m= missing one paired organ u= un. scheduled sac/ death 556 of 619 HISTOPATHOLOGY INCIDENCE TABLE GROUP  I

6 9 3 8 4

x

K

x h

h

x >

x h

h

E   1 6 9 3 9 s

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n n

n

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x x

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OVARY PITUITARY Hypertrophy, Pars Distalis SALIVARY GLAND, MANDIBULAR SKELETAL MUSCLE (RECTUS FEMORIS) Degeneration II 25 EPLj Experimental Pathology Laboratories, Inc. Key: X= Not Remarkable N= No Section I= lncomplete A= Autolysis l= minimal P= mild 3= rnoderate 4= swere P= Prsseni B= Benign M= Malignant m= mssing one paired organ u= unscheduled sac/ death 557of619 HISTOPATHOLOGY INCIDENCE TABLE WIL 417002 So Day Sacrifice Female Rat SKIN Inflammation, Preputial Gland SPINAL CORD, THORACIC SPLEEN Accessory Tissue Increased Hematopoiesis STOMACH Edema, Nonglandular Hyperplasia, Squamous Epithelium. Nonglandular Inflammation, Nonglandular THYMUS Congestion Necrosis, Cortical Lymphocytes THYROID TRACHEA Infiltrate, Mononuclear Cell Inflammation. Granulomatous Mineral Deposition URINARY BLADDER Infiltrate, Mononuclear Cell UTERUS Dilatation, Lumen EPL] II 26 6 6 9 9 3 3 f 5 6 4 6 x x x x x x x_ 2i XX x x x x x x X 1 x x x x 6 6 9 9 3 3 6 7 7 8 XX &_ LL x x x x x x &_ LL x x x x x x i x x x x GROUP 1

6 9 4 li H

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n n

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h     XX x x x x x_ zL X 3 2 xx x x X 1 x_ li X 3 E i Experimental Pathology Laboratories, Inc. Key: X= Noi Remarkable N No Section I incomplete A= Autolysis l= minimal P= mild ?.= moderate 4= swere P= Present B= Benign M= Malignant m= mtssing one paired organ u= unscheduled sac/ death 558 of 619 HISTOPATHOLOGY INCIDENCE TABLE GROUP

6 : 8 4 E

  1

6 9 3 5 4 !I                                        6 6 J i ; 6 6 6 7 x x t

6 9 3 ii ^           i 4

6 9 4 i h                                        WIL 417002 90 Day Sacrifice Female Rat VAGINA Inflammation I I I EPLj II 27 1 Experimental Pathology Laboratories, Inc. Key: X= Not Remarkable N= No Section I= lncomplete A= Autolysts l= minimal Z= mild 3= mcderate 4= severe P= Present B= Benign M= Malignant m= missing one paired organ u= unscheduled sac./ death 559 of 619 HISTOPATHOLOGY INCIDENCE TABLE GROUP WIL 417002 90 Day Sacrifice A Female Rat N I M A L ADRENAL Vacuolization, Cortex AORTA Inflammatory Cell Infiltrate, Adjacent Brown Fat BONE MARROW, STERNUM BONE, STERNUM BRAIN ESOPHAGUS EYE Hyperplasia, Cornea1 Epithelium Inflammation, Cornea Mineralization, Cornea Proteinaceous Fluid, Intraocular Retinal Atrophy Retinal Dysplasia HEART Myocardial Degeneration INTESTINE LARGE, CECUM Inflammation INTESTINE LARGE. COLON Nematode Parasite, Lumen

6 9 3 6 P

                   I I I I I I I I  I I I I I I I I I I 1 I I I 1 XI I I I I I I I I i I I I I I I I I I I I I I I I I IX/ I I I I I I 1 i t i 1 1 i i , I I I , I I I I I I / I I I I I I I I I I I I / I I I I I i i i i i i 1 i i i i i I 1 i i i. i i I I / I / I \ , ( I I I , I I I I I I I

I / I I I I I L EPLj II 28 Experimental Pathology Laboratories, Inc. Key: X= Not Remarkable N= No Section I= lncomplete A= Autoiysis l= minimal P= mild 3= rnoderale 4= severe P= Present B= Bemgn M= Malignant m= missing one paired organ u= unscheduled sac./ deaih 560 of 619 HISTOPATHOLOGY INCIDENCE TABLE GROUP 2 w1L 417002 90 Day Sacrifice Female Rat EPLl II 29 Experimental Pathology Laboratories. inc. Key: X= Not Remarkable N= No Section I= lncompleie A= Autolysis l= minimal P= mild 3= moderate 4= severe P= Present E=& nign M= Malignant m= missing one paired organ u= unscheduted sac./ death 561 of 619 HISTOPATHOLOGY INCIDENCE TABLE GROUP 2

6 9 3 6 z

i

z c

c

1 T

i

i

                                7 II WIL 417002 90 Day Sacrifice Female Rat i LIVER Degeneration/ Necrosis. Hepatocytes Hyperplasia, Bile Ducts Infiltrate, Mononuclear Cell Vacuolization, Hepatocytes LUNG W/ BRONCHI Alveolitisllnterstitial Pneumonia Hemorrhaqe LYMPH NODE, MANDIBULAR ConqestionlHemorrhaqe t t MAMMARY GLAND EPLj I I 30 Experimental Pathology Laboratories, Inc. Key: X Not Remarkable N No Section I= tncomplete A= AutolySis l= minimal Z= mild 3= moderate 4= swere P= Present B= Benign M Malignant m= missing one paired organ u= unscheduled %x/ death 562 of 619 HISTOPATHOLOGY INCIDENCE TABLE GROUP

6 9 3 8 2

i

z             r

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I I 4

I

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I

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6 9 4 0 9

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6 9 4 3 5

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6 9 4 1 3

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1

!I                                                                                WIL 417002 90 Day Sacrifice Female Rat A N M A L 6 6u 9 9 3 3 7 7 J37 1 1 2i__ L1_

t  T NASAL TURBINATE, LEVEL III Atrophy, Olfactory Epithelium Degeneration, Respiraton, Epithelium Infiltrate. Mononuclear Cell Inflammation Inflammation, Nasolacrimal Duct Inflammatory Cell Exudate, Medial Meatus Mineral Deposition VacuolizatiorXyst, Otfactory Epithelium NASAL TURBINATE. LEVEL IV Atrophy, Olfactory Epithelium Infiltrate, Mononuclear Cell Inflammation, Nasolacrimal Duct Mineral Deposition Respiratory Epithelial Metaplasia, Olfactory Epithelium Squamous Metaplasia, Respiratory Epithelium Vacuolization/ Cyst, Olfactory Epithelium t 1 1 Ii 31 EPLl Experimental Pathology Laboratories, Inc. Key: X Not Remarkable N= No Section I= lncomplete A= Autolysis l= minimal Z= mild 3= moderate 4= swere P= Present B= Benign M= Malignant m= missing one paired organ u= unscheduled sac./ death 563 of 619 HISTOPATHOLOGY INCIDENCE TABLE GROUP WIL 417002 SO Day Sacrifice Female Rat A N il A L EPL( II 32 Experimental Pathology Laboratories, Inc. Key: X= Not Remarkable N= No Section I= lncomplete A= Autolysis l= minimal P= mild 3= moderate 4= severe P= Present B= Benign M= Malignant m= missing one paired organ u= unschedukd sac./ death 564 of 619 HISTOPATHOLOGY INCIDENCE TABLE WIL 417002 90 Day Sacrifice Female Rat GROUP 2 EPLl II 33 Experimental Pathdogy Laboratories, Inc. Key: X= Nol Remarkable N= No Section I= lncomplete A= Autolysis l= minimal ?= mild a= moderate 4= severe P= Present B= Benign M= Malignant m= missing one paired organ u= unscheduled sac/ death 565 of 619 HISTOPATHOLOGY INCIDENCE TABLE WIL 417002 90 Day Sacrifice Female Rat A N I M 6 6 6u A 9 99 L 3 33 6 77 2 67 UTERUS X Dilatation, Lumen VAGINA Inflammation 1 / I I I I , I I I I I I I I I I 1 I I I I I I I I EPLl Experimental Pathology Laboratories, Inc. GROUP +

II 34 I I I I I I I I I I / I / I I I I I I I I I / I I I I I I I I I I Key: X= Not Remarkable N= No Section I= lncomplete A= Autolysis l= minimal Z= mIld 3= moderate 4= severe P= Present B= Benign M Malignant m= mlssing one paired organ u= unscheduled sxideath 566 of 619 HISTOPATHOLOGY INCIDENCE TABLE GROUP r 5 I '

E 4 2 3i

r

E

i z

1

z   1

I z    WIL 417002 go Day Sacrifice A Female Rat N I M 6 A 9 L 3 : KIDNEY inflammation, Pelvis Mineralization 1 Nephropathy 1 LARYNX, LEVEL I Infiltrate, Mononuclear Cell 1 inflammation, Granulomatous Mineral Deposition 6 6 9 9 3 3 8 9 a J 1 1 Cl 1 1 1_ J _ 1 1 1_ 1 1 1 1 1 'tx 6 6 9 9 4 4 2 3 83 1 1 N Ll 1 21 1 1 2 1 1 1 1 1 1 1 1 x x . L LARYNX, LEVEL II Hyperplasia, Transitional Epithelium Infiltrate, Mononuclear Cell Inflammation, Granulomatous 1 inflammatory Cell Exudate, Lumen Mineral Deposition 1 Squamous Metaplasia, Transitional Epithelium I i i i i i i 1 i i i i i i I i i i i i i EPL( II 35 X= Nol Remarkable N= No Section klncompkte A= Autolysis t= minimal Z= mild 3= moderate 4= swew P= Present B= Eenign M= Mallgnant m= missing one paired organ u= unscheduled %x./ death Experimental Pathology Laboratories, Inc. Key:

567 of 619 HISTOPATHOLOGY INCIDENCE TABLE GROUP WIL 417002 90 Day Sacrifice Female Rat A N M A L NASAL TURBINATE, LEVEL I Hyperplasia, Squamous Epithelium Hyperplasia, Transitional Epithelium Infiltrate, Mononuclear Cell inflammation Inflammation. Granulomatous Inflammatory Cell Exudate, Medial Meatus NASAL TURBINATE, LEVEL II Infiltrate, Mononuclear Ceil Inflammation, Nasolacrimal Duct Inflammatory Cell Exudate, Medial Meatus Mineral Deposition Vacuolization/ Cyst, Olfactory Epithelium 3 I I I I I I I I I I II I I 6666666666 9999999999 3333334444 5678892233 1 i i i EPLI II 36 Experimental Pathology Laboratories, Inc. Key: X= Not Remarkable N= No Section I= lncomplete A= Autolysis l= minimal P= mild 3= moderate 4= severe P= Present B= Benign M MalIgnant m= missing one paired organ u unscheduled sac/ death 568 of 619 HISTOPATHOLOGY INCIDENCE TABLE GROUP WIL 427002 go Day Sacrifice Female Rat A N M A L NASAL TURBINATE. LEVEL VI X Atrophy, Olfactory Epithelium Infiltrate, Mononuclear Cell Mineral Deposition TRACHEA X Infiltrate, Mononuclear Ceil Inflammation, Granulomatous Mineral Deposition EPL( II 37 7 6 9 3 9 4

I

1

!I h

6 9 4 2 s

2

3

I

f H

6 9 4 2 s

z

1

z zc            t Experimental Pathology Laboratories, Inc. Key: X= Not Remarkable N= No Section I= lncomplete A= Autolys~

l= minimal 2= mild B= moderate 4= severe P= Present B= Benign M= Malignant m= mising one paired organ u= unscheduk? d sac. ldeath 569 of 619 HISTOPATHOLOGY INCIDENCE TABLE GROUP WIL 417002 90 Day Sacrifice Female Rat ADRENAL Vacuolization, Cortex AORTA inflammatory Cell Infiltrate, Adiacent Brown Fat BONE MARROW, STERNUM BONE, STERNUM BRAIN ,ESOPHAGUS EYE Hyperplasia, Cornea1 Epithelium Inflammation, Cornea Mineralization, Cornea Proteinaceous Fluid, Intraocular Retinal Atrophy Retinal Dysplasia INTESTINE LARGE, CECUM Inflammation INTESTINE LARGE, COLON Nematode Parasite, Lumen EPLj II 38 6 6 9 9 3 3 7 7 5 9 x x f 4 i : 4 4 0 0 18 XX xx x x x x x x XX X 1_

X 1 1 1 XX x x 6 6 9 9 4 4 1 2 57 x x xx x x x x xx x x LL x x 1 1 x x x x

6 9 4 3 1 H

x

x z x x E

h

1 x

4     I

Experimental Pathology Laboratories, Inc. Key: X= Not Remarkable N= No Section I= lncomplete A= Autolysis l= minimal Z= mild 3= moderate 4= severe P= Present B= Benign M= Malignant m= missmg one paired organ u= unscheduk? d sacldeath 570 of 619 HISTOPATHOLOGY INCIDENCE TABLE WIL 417002 90 Day Sacrifice Female Rat I I I / IPIPI I t III I I I I I I I I II I II III/ LARYNX, LEVEL II I IX/ 1 II 1) / / / 1 j / 11 ) / / 4acia Tmncitinnal III I I I I I I I I III I I I I I I Hype& .__._, . ._.... ._. Epithelium t I I I I 11121 II III Ill 121 Infiltrate, Mononuclear Cell I inflammation, Granulomatous ,2,: ,. , /., Inflammatory Cell Exudate, 1 Lumen EPLl II 39 Experimental Pathology Laboratories, Inc. Key: X= Not Remarkable N= No Section l= lncomplete A= Autolysis l= minimal Z= mild B= moderate 4= severe P= Present 8= 8enign M= Malignani m= missing one paired organ u= unscheduled sacldeath 571 of 619 HISTOPATHOLOGY INCIDENCE TABLE WIL 417002 90 Day Sacrifice Female Rat A N I M 6 6 6 A 9 9 9 L 3 3 3 7 7 8 LUNG W/ BRONCHI Alveolitis/ lnterstitial Pneumonia Hemorrhage x x x I LYMPH NODE, MEDIASTINAL ixjxlx Congestion/ Hemorrhage LYMPH NODE, TRACHEOBRONCHIAL X X X ConoestionlHemorrhage MAMMARY GLAND x x x NASAL TURBINATE, LEVEL I x x x Medial Meatus I I I NASAL TURBINATE, LEVEL II X Infiltrate, Mononuclear Cell inflammation, Nasolacrimal nt1ct . Inflammatow Cell Exudate, Medial Meatus Mineral Deposition 1 1 VacuolizationKyst, Olfactory Epithelium EPL(

Experimental Pathology Laboratories, Inc. GROUP  r+% 7

6 9 4 2 1

i

1 x

1 h

L(_

x

I II 40 Key: X= Not Remarkable N= No Section I= lncomplete A= Autolysis l= minimal P= mild 3= moderate 4= severe P= Present B= Benign M= Malfgnant m= missing one paired organ u= unscheduled sac./ death 572 of 619  HISTOPATHOLOGY INCIDENCE TABLE WIL 417002 GROUP 4

90 Day Sacrifice Female Rat Epithelium NASAL TURBINATE, LEVEL V Atrophy, Olfactory Epithelium Infiltrate, Mononuclear Cell Inflammatory Cell Exudate, Medial Meatus Mineral Deposition Respiratory Epithelial Metaplasia, Olfactory Epithelium VacuolizatiorXyst, Olfactory Epithelium EPL/ II 41 6 9 4 2 1

I

1

4

1       i 6 9 4 3 r zc

I

r

z

z

x

                                         T 1 Experimental Pathology Laboratories. Inc. Key: X= Not Remarkable N= No Section I= tncomplete A= Autolysis t= miniinat Psmitd S= moderate rl= severe P= PreseW B= Benign M= Mahgnant m= missing one paired organ wunscheduled sac/ death 5'73 of 619 HISTOPATHOLOGY INCIDENCE TABLE WIL 417002 90 Day Sacrifice Female Rat NASAL TURBINATE, LEVEL VI Atrophy, Olfactory Epithelium Infiltrate, Mononuclear Cell Mineral Deposition OPTIC NERVE Degeneration, Vacuolar OVARY OVIDUCT Inflammatory Cell Exudate, Lumen PANCREAS Atrophy, Acinar Cell infiltrate, Mononuclear Cell PARATHYROID PERIPHERAL NERVE (SCIATIC)

PITUITARY Hypertrophy, Pars Distalis SALIVARY GLAND, MANDIBULAR SKELETAL MUSCLE (RECTUS FEMORIS) Degeneration SKIN Inflammation, Preputial Gland SPINAL CORD, CERVICAL SPINAL CORD, LUMBAR Hemorrhage Myelomalacia EPL\ GROUP T II 42

  1 /

1 X I I I :m/ I I I I I I / I I I I I I I I I I , / I I I I I I Experimental Pathology Laboratories, Inc. Key: X= Not Remarkable N= No Section I= lncomplete A= Autolysis l= minimai *= mild 3= moderate 4= severe P= Present B= Benign M= Malignant m= missing one pared organ u= unscheduled sac/ death 574 of 619 HISTOPATHOLOGY INCIDENCE TABLE GROUP 4

                                    1 WIL 417002 go Day Sacrifice Female Rat 9" : 4 4 1 2 5 7 li_ X x x x x x x xx x 1 K x I( x I( x SPINAL CORD, THORACIC SPLEEN Accesson/ Tissue Increased Hematopoiesis x x x x 1 x x x x +

THYROID X 1 X 2 1 x x URINARY BLADDER Infiltrate, Mononuclear Cell UTERUS Dilatation, Lumen VAGINA Inflammation I I I I I I I I I / I I I I I / / I I ,

I Experimental Pathology Laboratories, Inc. Key: X= Nol Remarkable N= No Section I= lncompkte A= Autolysis l= minimal Z= mild 3= moderate 4severe P= Presenl B= Bemgn M= Malignant m= missing one pared organ u= unscheduled sac./ death 575 of 619 CORRELATION OF GROSS AND MICROSCOPIC FINDINGS 576 of 619 WIL 417002 90 Day Sacrifice Species: Rat Animal Number 3308 3314 CORRELATION OF GROSS AND MICROSCOPIC FINDINGS SW Males Group Identification: 1  Sacrificed Client Topoarauhv / Site Client Gross Observations UNGS Area( s), white, few, irregularly shaped, all lobes YMPH NODE, TRACHEOBRONCHIAL Discoloration, dark red YMPH NODE, MEDIASTINAL Discoloration, dark red KIN Matting, red, ocular, left KIN Matting, red, ocular, left L EPLl Experimental Pathology Laboratories, Inc. 111 1 Microscopic Observations Jo Correlatin V/ BRONCHI 7 Lesion (LUNG ZongestionlHemorrhage ZongestionlHemorrhage Jo Comment Required 40 Comment Required CORRELATION OF GROSS AND MICROSCOPIC FINDINGS WIL 417002 90 Day Sacrifice Species: Rat Animal Number 69305 69320 Client l` ouoeranhv / Site YMPH NODE, MEDIASTINAL Sex:

.UNGS Client Gross Observations Discoloration, dark red Males Group Identification:

Area( s), white, few, irregularly I shaped, all lobes F E `ANCREAS ;KIN ;EMlNAL VESICLES Pale Matting, red, ocular, left Small, right 2  Sacrificed I ;

69338 69347 I No Correlating Lesion No Comment Required Reduced Secretion (SEMINAL VESICLE)

Experimental Pathology Laboratories, Inc. III 2 CORRELATION OF GROSS AND MlCROSCOPlk FINDINGS WIL 417002 90 Day Sacrifice Species: Rat S& X: Males Group Identification: 3  Sacrificed Animal Number 3328

3348 f s Client Topoeraphy / Site PLEEN TOMACH f P EPL(

Experimental Pathology Laboratories, Inc. Client Gross Observations rccessoiy, one, 3 mm in diameter iontents, dark red

Ill 3 Microscopic Observations iccessory Tissue Jo Correlating Lesion WIL 417002 QO Day Sacrifice Species: Rat Animal Number 9304 9307 9329 i9346 EPLl CORRELATION OF GROSS AND MICROSCOPIC FINDINGS Sex: Males Group Identification: 4  Sacrificed `HYMUS Client Toooeraphv / Site UNGS KIN KIN :YES/ OPTIC NERVES Client Gross Observations discoloration, dark red ,rea( s), white, few, irregularly haped, all lobes latting, red, ocular, right ,latting, red, ocular, right iye( opacity, left longestion longestion MicroscoDic Obsewations MicroscoDic Obsewations $~$~$ tiicj Lesion (LUNG $~$~$ tiicj Lesion (LUNG Jo Comment Required Jo Comment Required Vo Comment Required Vo Comment Required :hE) :hE) i perplasia, Cornea1 Epithelium i perplasia, Cornea1 Epithelium Experimental Pathology Laboratories, Inc. CORRELATION OF GROSS AND MICROSCOPIC FINDINGS WIL 417002 90 Day Sacrifice Species: Rat Sex: Females Group Identification: Animal Number 69367 Client Touoeraphv / Site EYES/ OPTIC NERVES Client Gross Observations Eye( s)  opacity, right LYMPH NODE, MANDIBULAR Discoloration, dark red, right EPL III 5 Experimental Pathology Laboratories, Inc. 1  Sacrificed Microscopic Observations Inflammation, Cornea (EYE)

Congestion/ Hemorrhage CORRELATION OF GROSS AND MICROSCOPIC FINDINGS WIL 417002 SO Day Sacrifice Species: Rat Animal Number 69362 69390 69396 Sex: Females Group Identification:

Client Touoarauhy / Site YMPH NODE, MANDIBULAR JJNGS SKIN `ITUITARY SKELETAL MUSCLE EPL]

Experimental Pathology Laboratories, Inc. Client Gross Observations Discoloration, dark red, bilateral 4rea( s), white, few, irregularly shaped, all lobes flatting, yellow, urogenital small liscoloration, dark red, rurroundin mechanical trauma frr; ture ofgtibia and ftbula,

Ill 6 2  Sacrificed Microscopic Observations Congestion/ Hemorrhage g;; rl$ tn{ Lesion (LUNG No Comment Required No Correlating Lesion Hemorrhage (SKELETAL MUSCLE, OTHER) CORRELATION OF GROSS AND MICROSCOPIC FINDINGS WIL 417002 OO Day Sacrifice Species: Rat SW Animal Number 69377 Client Topography/ Site INTESTINE SMALL, JEJUNUM LIVER LUNGS SKIN EPL(

Experimental Pathology Laboratories, Inc. / Females Group Identification:

Client Gross Observations Contents, dark red Pale, all lobes Area( s), dark red, few, irregularly shaped, right diaphragmatic lobe Matting, red, ocular, bilateral Contents, dark red I 2  Unscheduled Sac/ Death No Correlating Microscodc Observations Lesion Vacuolization, Hepatocytes Hemorrhage (LUNG W/ BRONCHI)

No Comment Required No Correlating Lesion III 7 CORRELATION OF GROSS AND MICROSCOPIC FINDINGS WIL 417002 QO Day Sacrifice Species: Rat Animal Number 69375 69379 69383 69415 69427 SKIN Client Touoaraphv / Site Client Gross Observatiotk Matting, yellow, urogenital LIVER Area( s), raised, one, 3 x 3 x 1 mm, white, median lobe LUNGS Area( s), white, few, irregularly shaped, all lobes OVARIES Area( s), white, multiple, pinpoint, bilateral SKIN Matting, yellow, urogenital SKIN Matting, yellow, urogenital; anogenital JTERUS Contents, clear fluid, both horns 69431 I S& X: Females Grollp Identification: I EPLl Experimental Pathology Laboratories, Inc. ill 8 4  Sacrificed Microscopic Observations No Comment Required No Correlating Lesion No Correlating Lesion (OVARY)

No Comment Required No Comment Required Dilatation, Lumen WIL 4 17002 HAP Task Force 1, 1,2 Trichloroethane APPENDIX L Study Protocol 585 of619 Sponsor: HAP Task Force A. Title of Studv:

A 90 Day Inhalation Toxicity Study of 1, 1, Z Trichloroethane (I, I, 2 TCE) in Rats (With Satellite Groups for Pharmacokinetic Evaluations in Rats and Mice)

B. Protocol Modification:

1) VIII. Experimenta Observations:

The following information modifies that currently in the protocol.

H. Anatomic Pathology:

3. Microscopic Examination (Toxicology Groups):

All tasks for histological preparation and microscopic examination of tissues will be performed by Experimental Pathology Laboratories, Inc. (EPL). The study pathologist will be Frederick A. Ruecker, D. V. M., M. S., D. A. C. V. P. EPL will provide an audited pathology report for inclusion in the study report. EPL will be responsible for all Quality Assurance activities for these tasks.

C. Reason for Protocol Modification:

To modify Anatomic Pathology section to state that histology and microscopic examination will be performed by Experimental Pathology Laboratories, Inc.

Approved By:

HAP Task Force ' Peter Vo$ tek< Ph. D. Datk Sponsor Representative Prepared By:

WIL Research Laboratories, Inc. ,

Study Dir& r WIL R!? X. EARCH LABOR.% TORIES. JNC 1 107 GEORGE ROAD ASHLAND. OH 4 18o59281 1419) 2848100 F. A?( (~ IQIZ~ Q~

586 of 619 Study Number: WL 417002 PROTOCOL AMENDMENT II Sponsor: HAP Task Force A. Title of Studv:

A 90 Day Inhalation Toxicity Study of 1, I . 2 Trichloroethane (1, 1, I TCE) in Rats (With Satellite Groups for Pharmacokinetic Evaluations)

B. Protocol Modifications:

1) V. Test Svstem:

The following information modifies that currently in the protocol:

C. Source: Charles River Laboratories G. Identification System: Effective May 21. 2001, the cage label will not include the date of animal arrival or the date of initiation of exposures.

VII. Experimental Desk:

3 A. Animal Receipt and Acchmation:

3) E. The following information adds to that currently in the protocol:

To allow suitable evaluation of animals for group assignment. the animals (rats and mice) to be used for randomization for the pharmacokinetic evaluation groups will receive a detailed physical examination within 3 days of the day of group assignment.

Exposure Methods:

The following information adds to that currently in the protocol and will be effective May 3 1,200 1.

The exposures will be conducted in 2 m' stainless steel and glass whole body exposure chambers. These chambers will be located in study room 51.

587 of 619 WlL~ 17002 Protocol Amendment II Page Two Animal cage batteries will be rotated on a daily basis through various chamber locations in order to reduce the potential differences in exposure due to any residual non homogeneity in chamber concentrations. The rotation procedure to be used will be described in the data records.

Drinking water will be avaitable to mice during exposures.

On each day of PK blood collection (1': 3ti and 5" days of exposure of the fourth exposure week), the female rats designated for blood collection on the specific day will, be exposed to the target concentration of 100 ppm in a 500 L NYU type whole body chamber (that is, separate from the remaining animals in the group). This will be a completeiy separate exposure system with its own test atmosphere vapor generator, chamber dilution airflow and calibrated exhaust airflow. The methods used for this generation and exposure system will be similar to those for the other test article systems. Specific details, settings and parameters for this system, which wiIl be used on three days only, will be documented in the study records. The exposure chamber will be monitored using the same methods and according to the same schedule as for the other exposure chambers. The 12 animals scheduled for blood collection on the specific day will be placed in a Ii compartment exposure battery, which will then be placed in the exposure chamber. Only one exposure battery placement level will be used for the three days this chamber is used. This change for the pharmacokinetic (PK) study rats will facilitate blood collection at the appropriate time points relative to the start of exposure and prompt collection of blood after animals are removed from the exposure chamber (4 and 6 hour time points). The use of different exposure systems for PK study rats and mice will permit the PK rat exposure to be started approximately 30 minutes after or before the mouse exposure.

4) F. Test Atmosphere Generation Methods:

Should be modified as follows. and will be effective on May 31. X01.

Vapors of the test article will be generated using a glass bead vaporization system similar to that shown in Figure 1 and Figure 2. The vaporization column consists of a glass tube filled with glass beads. For Groups 2.3 and 4, the column will be 3.4 cm in diameter (ID). 75 cm long filled with 6 and 12 mm beads. Calibrated Harvard Syringe Pumps (model 97.5 with a 20 cc syringe for Group 2 and model 975 with a 50 cc syringe for Group 3) and a calibrated FMT pump (model QG 6 with a l/ S inch piston for Group 4) will be used to meter test article to the column.

j:, r .

1: ,.:.,_ .;: ..;:: .:. i, ._:: _:,_ i:::::: .: ..:. _ .; `ipi / iv.; ./. j__ .

.._ ;..::. : .. i f _ : 5, ; i.

!a? a§ s 588 of 619 wlL 41700~ Protocol Amendment II Page Three Compressed air will be metered to the bottom of the column using calibrated flowmeters. Vaporization occurs as the test article flows down through the beads and the compressed air flows up through the column. The concentrated vapors are piped to the chamber inlet where the concentration is reduced to the desired level by mixing with the chamber ventilation air. The vaporization columns are heated by heating rapes operated from Omega CN370 temperature controllers.

The following table indicates the specific generation conditions for each exposure group.

Bead Flow Rate of Test Article Column Compressed Air Chamber Metering Flow Rate Temperature for Vaporization Number 3ciairz 0 422 1 NA NA NA 75 17 2 Harvard 20 ind. 0.038 75 18 3 Harvard 20 ind. 0.080 75 19 4 l 341 30 4092 full scale 0.186 0.242 75 17 Nominal Chamber Ventilation  430  430  430  430 Chamber ventilation flow rate may be varied within 400 to 500 LPM to maintain the desired exposure concentrations; any other changes to these operational parameters will be authorized by the Inhalation Department Director, Study Director or appropriate designee and recorded in the study records.

3 G. Methods for Characterization of Exposure Atmospheres:

Sections G. l 2 should be modified as follows and will be effective as of May 31, ZOOl:

1. Nominal Concentrations:

Nominal exposure concentrations will be determined by weighing the containers of test article prior to and again after each day's exposure, and calculating the concentration from the weight difference and the total volume of air passed through each chamber during that day's exposure.

:.. .:_ .~, .:: i ,2,$ .. `i: _::: : _:.: ,~:.:;::,_;:: g: i::. :` ilix:. ie; j~ ii; i ii"`; / . . . . . . .,,:. . : _; :_ : ::: : i ~/ . . . . `( i i iz? cmpy `i 589 of 619 . w& 41700:! Protocol Amendment II Page Four 2. Actual Exposure Concentrations:

Actual exposure concentrations will be determined approximately every 35 minutes using a gas chromatographic method.

The following table summarizes the operational conditions of the gas chromatograph and signal integrator:

GC Manufacturer Model No. Hewlett Packard 5890 Series II Detector Type Detector Temperature Column Diameter X Length Column Type Fuel Gas Fuel Pressure Fuel Flowwte Air Pressure Air Flowrate Carrier Gas Carrier Gas Pressure Carrier Gas Flowrate Injection Volume Retention Time Integrator Manufacturer Model Number Chart Zero Offset Chart Attenuation Chart Speed Peak Area Rejection Value Peak Threshold Peak Width FID 250%

0.25 mm x 30 m DB WAX Hydrogen 15 psig 30 mllmin.

25 psig 300 mVmin.

Helium SO psig 10 ml/ min 0.35 ml Approx 0.825 min.

Hewlett Packard 5890 0 1 1 .O cmimin. 0 2 .ii.. i:. :: `7. ii :: J/ i, pi:;:; i;; ijhG ::;. :::...~:~ ::.:.. y::., rYe"` i; j/:.: i .,_:,: . .:___ ' ii L i, /j ; :: :: E : ::, :,

ki? zmB'

590 of 619 wIL 417002 Protocol Amendment II Page Five Any changes to these operational parameters will be authorized by the Inhalation Department Director, Study Director or appropriate designee and recorded in the study records.

Using the GC and integrator, samples will be collected manually and the integrator will operate a gas sampling valve with a 0. X ml sample loop, display the chromatogram and calculate the area under the sample peak. The operator then uses a slope and intercept, from the GC calibration curve, to calculate the measured concentration in patts per million (ppm).

The GC will be calibrated using standards prepared in Ted@ gas bags. The gas bag standards will be prepared as follows: A known volume of test atticle will be injected into a heated (80° C) glass vaporization bulb. Compressed air will be metered into the bulb to vaporize and dilute the test article. The total volume of compressed air used will be measured with a dry test meter. The resulting vapors will be collected in 40 liter gas bags.

Prior to initiation of animal exposures, the instrument will be calibrated with sets of five standards prepared in triplicate. The calibration curves will be prepared and defined by 9,27,72, 109 and 136 ppm standards. A least squares hne will be fitted to the data set for each set of standards to provide a prime standard curve. Thereafter, once each day and prior to initiation of exposures, a calibration check will be performed using a single freshiy prepared standard. This standard will be one of the five vahtes used for prime calibration, and each day a different one will be used. If the daily calibration check is within 210% of the prime calibration curve, the instrument will be considered to be within normal calibration limits. The following table summarizes the standard Standard Volume of Concentration Test Article 0 fdJ 9 1.0 80  2.4 30 27 3.0 80  2.4 30 72 8.0 80  2.4 30 109 12.0 80  2.4 30 136 15.0 80  2.4 30 Nominal Vaporization Temperature 0 Nominal Vaporization Flow Rate ILPM) Total Dilution Air Volume i& J _::: .:: _ ::_ :::. ::.. :: ..:::., .::. :; _i, i::::,; .:.. ::::::, .:I ;;: iii;:: k<; ;(;? z %: ,, .:,. i :. :: : 1:. : j: :: 7.: /::, iz rilmsma'

591 of 619 . wiL417002 Protocol Amendment Il Page Six VIII. Exnerimental Observations:

6) B. CIinical Observations (All Animals):

7) C. The following information modifies and/ or clarifies that currently in the protocol.

For toxicology evaluation animals: On exposure days, cIinica1 observations will be performed twice per day, prior to exposure and following exposure (O 2 hours following end of exposure). On non exposure days, clinical observations will be performed once in the morning. For pharmacokinetic evaluation animals: On exposure days, clinical observations will be performed once, prior to exposure. On non exposure days, clinical observations will be performed once in the morning.

Detailed Physical Examinations (Toxicology Groups):

The following information clarifies that currently in the protocol.

All animals will receive a detailed physical examination at least one week prior to and on the day of group assignment, weekly during the exposure phase, and on the day following final exposure (prior to necropsy).

8) The foIlowing information clarifies that currently in the protocol (VIII. D and E).

D. Individual Body Weights (All Animals):

Individual body weights will be recorded weekly, beginning at least one week prior to test material administration. For the toxicology group animals, a fasted weight will be determined prior to blood collection and necropsy.

E. Individual Food Consumption (Toxicology Groups):

Individual food consumption will be recorded weekly for all animals, beginning at least one week prior to test material administration.

592 of 619 wIL 417002 Protocol Amendment II Page Seven 9) H. Anatomic Pathology:

1. Macroscopic Examination (Toxicology Groups):

The following information clarifies that currently in the protocol.

Tissue List: Items shouid be Exorbital lacrimal glands (2) and Parathyroid. d Footnote describes requirements for microscopic examination of parathyroids.

10) 1. Blood Collection and Processing for Pharmacokinetic Groups:

The following information adds to and/ or clarifies that currently in the protocol.

To accommodate the Chour time point, technicians equipped with appropriate protective equipment. including Tyvek' coveralls and supplied air respirator. will quickly open the high concentration chambers (during exposure), remove designated animals (3 rats and 3 mice) and then reseal the chambers. The same procedure wilt be used for the &hour time point, except the generator will be turned off at 6 hours.

As quickly as possible, the animals scheduled for blood collection at a specific time point (and day) wiIl be anesthetized using isoflurane and blood will be collected from the vena cava into heparinized syringes. Blood will be transferred in duplicate directly into headspace vials for analysis, and remaining' bulk blood will be collected and saved in vacutainers (with dry sodium heparin). For the headspace vial samples. duplicate blood samples of 100 1.11 from each animal wilt be aliquoted into cold, pre weighed 20 ml headspace vials with crimp tops and PTFE silicone septa. The vials will be sealed as quickly as possible. The headspace vials will be weighed before and after the addition of blood to verify volumes added gravimetrically. Immediately after weighing, the vials will be placed on dry ice to freeze the biood samples. For each animal, the remaining blood will be placed in a pre weighed, pre cooled vacutainer tube, which will be sealed and re weighed. Dry ice will also be used to freeze this extra blood. At the time of sacrifice, storage stability spike standards will be prepared in headspace vials using control blood (from untreated rats) spiked with known quantities of 1,1.2 trichloroethane. in addition, unspiked control bIood (from untreated rats) will be frozen and shipped to the analytical laboratory, Battelle Northwest Laboratories, for preparation of standards (for calibration curves) and samples of 1,12 trichloroethane solution( s) used to prepare the storage spike samples will be included in the shipments. :::: .::. ::: .:._ _:_ :::: `7' .*::. li ..::... if: y,: jiiii::: ii: e; y;; ; `+$ ; .: :: :i + : "%

593 of 619 WL417002 Protocol Amendment Ii Page Eight Samples will be shipped by next day service in Styrofoam containers on dry ice in order to be received at Battelle on a Tuesday, Wednesday or Thursday. Samples may be stored at 70° C until shipment, if necessary. The blood samples and other samples described above will be shipped to the following contact person and address:

Attn: Torka Poet, Ph. D. Bldg. 331, Lab 313: P7 59 Battelle Warehouse 790 6th St. Richland, WA 99352 The analytical laboratory and/ or the Sponsor will be responsible for all quality assurance activities or requirements related to the sample analyses. However, copies of quality assurance inspection repotts will be supplied to the study director at WIL for review and evaluation of potential effects on study outcome, quality and integrity. The analytical Iaboratoty and/ or the Sponsor will also be responsible for subsequent data analysis and reporting for the PK evaluations.

The lungs, liver, kidneys, spleen and whole brain will be collected at the time of blood collection and stored at 70° C by WIL Research Laboratories, Inc. for up to 2 years. To minimize chemical loss, all sample manipulations will be conducted on ice (to maximum extent possible). Each organ will be double wrapped in plastic wrap, placed in a labeled aluminum foil pouch, frozen in liquid nitrogen and then sealed in a plastic storage bag.

C. Reasons for Protocol Modifications:

1) To modify information regarding the study animals.

2) To clarify requirements for detailed physicals prior to group assignment for the PK evaluation animals.

3) To add additional information on exposure methods.

4) To define test atmosphere generation methods.

5) To define exposure atmosphere characterization methods.

6) To clarify daily schedules for clinical observations.

7) To clarify the schedule for detailed physicals of toxicology group animals. ;: h .3: ;: i: ,:: i; i : :: i' :: i cc::: xi':;: p:;: iii yz;. .::: i.:: 7: g:; / ., J. ? .:.. ;. y :, :: B (' .: i LI 7 :: ;;. i. `< RiPc? ak.

594 of 619  WJ. L 417002 Protocol Amendment II. Pap Nine 8) To clarify schedules for body weights and food consumption measurements.

9) To correct information in tissue list.

10) To clarify and add information for the pharmacokinetic evaluations.

Approved By:

HAP Task Force Peter Voytec Ph. D. Sponsor Representative Prepared By:

WIL Research Laboratories, Inc.

Study Director 595 of 619 WIL 417002 Protocol Amendment II Page Ten Figure 1: Schematic Diagram of Exposure and Generation System Groups 2 & 3 source of Ii c Chamber Supply _I_ Air iI 20004W H~ MCHI Chamber j Havard I Pump i i b Glass Bead Column 8 iemperature TO controller Compissed Air Source * Orifice Meter

To Chambw Exhaust 596 of 619 wlL 417002 Protocol Amendment II Page Eleven Figure 2: Schematic Diagram of Exposure and Generation System Group 4 source of I !  Chamber Supply ii Ait 2OtXWtar Hazleton Chamber I t > ' \i Glass Bead i : i Column& Temwature TO Controller Test Material Compressed Air Source

94%  v Orifice Meter

7 To Chamber Exhaust =?` I. .. ' ;:: _?:. y :I : Y :: .A.,, .c ,.: i: .. i._ .::::::;._:I ::::.::. :::I  .::: 1: ! 2, :::::: / .: p,: ytr ;y : .i.~ . U.: Y i I :! 7 +? / :: ;j: 1: i;

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597 of 619 Study Number: WlL 417002 PROTOCOL AMENDMENT I Sponsor: HAP Task Force A. Title of Studv:

A go Day inhalation Toxicity Study of 1, 1, 2 Trichloroethane (I, 1, 2 TCE) in Rats (With Satellite Groups for Pharmacokinetic Evaluations)

B. Protocol Modifications:

Title of Studv:

1) The following modifies the study title as required by WIL JACUC.

A 90 Day Inhalation Toxicity Study of 1, 1. 2 Trichloroethane (1, 1, ?, TCE) in Rats (With Satellite Groups for Pharmacoknetic Evaluations in Rats and Mice)

III. Studv Schedule:

The following information replaces that currently in the protocol.

Proposed Experimental Termination Date: To be added when dates of microscopic examinations are known Proposed Audi ted Draft Report Date:

Proposed Final Report Date: December 15.200 1 February 1.5, XK!

598 of 619 wL417002 Protocol Amendment I Page Two VII. Experimental Design:

3) D. Organization of Test Groups, Exposure Levels and Treatment Regimen:

1. Organization of Test Groups:

The following table presents the study group arrangement.

Groups 5 and 6 will be exposed using the same exposure system and methods used for Group 4. Data for the toxicokinetic groups will be collected in separate WTDMS' computer protocols (from the toxicology groups).

C. Reasons for Protocol Modifications:

1) To modify study title to include species for pharmacokinetic evaluations as required by WrL IACUC. wIL 417001 Protocol Amendment I Page Three 3 To modify information for study schedule.

3) To clarify target exposure concentrations.

Approved By:

HAP Task Force feL d hii k /w Pete: Voytek, Ph. D. Sponsor Representative Date Prepared By:

WIL Research Laboratories, Inc.

Study Director .~, :+ c? =:, i. :i. ..:: i.. 2:. A.. zi; +; zii;;; i<. 2::::: ._ ix::. i ::: :: s.:: "` 5 :i': i : .&. :`. g' : L $is. Caiij f, /5 600 of 619 PagclDf19 PROTOCOL WIL 417002 May 15.2001 A !& DAY INHALATION TOXICITY STUDY OF 1, 1, ZTRICHLOROETHANE (1, 1, Z TCE) IN RATS (WITH SATELLITE GROUPS FOR PHARMACOKINETIC EVALUATIONS)

(U. S. EPA TSCA 799.9346 Guideline)

Submitted To:

HAP Task Force do Peter Voytek, Ph. D. P. O. Box 331 Millwood. VA 23646 WIL Research Laboratories, Inc. 1407 George Road Ashland, OH 44805 9181 601 of 619 w1L 017002 Rp2of 19 May IS. 2001 I. Obiective:

II. The objective of this study is to evaluate potential subchronic toxic effects of the test article when administered via inhalation to rats for 90 days. In addition. 36 female F 344 rats and 36 female B6C3Fl mice will be exposed by inhalation for 28 days and used for a pharmacoklnetic study.

This protocol has been designed to generally conform with the U. S. Environmental Protection Agency (EPA) TSCA guideline 799.9346: 90 Day Inhalation Toxicity Study.

This study will be conducted in compliance with the Good Laboratory Practices Regulations of the U. S. Environmental Protection Agency  TSCA: 40 CFR Part 792.

Personnel Involved in the Studv:

A. Sponsor Representative:

Peter Voytek, Ph. D.

B. WIL Study Director:

Daniel T. Kirkpatrick, Ph. D., D. A. B. T. Assistant Director, Inhalation Toxicology C. WIL Deputy Director:

Christopher P. Chengelis. Ph. D., D. A. B. T. Director, Toxicology D. WIL Departmental Responsibilities:

Jozef J. W. M. Mertens, Ph. D.. D. A. B. T. Assistant Director, Toxicology Gary R. Kiplinger, B. S. Manager II, Inhalation Ronald E. Wilson, B. S. Director, Informational Systems 602 of 619 WIL 417@ 3? Pdgc30fl9 May 15.2coI Carol A. Kopp, B. S. Group Supervisor, Gross Pathology and Developmental Toxicology Laboratory Jane M. Nagel, M. B. A., B. S., MT( ASCP) Manager, Clinical Pathology Mike Safron, A. S., HT( ASCP) Manager, Histology Sally A. Keets, A. S. Manager II, Vivarium Deborah L. Little, B. S., RQAP GLP Manager II, Quality Assurance Daniel W. Sved, Ph. D. Director, Metabolism and Analytical Chemistry Charlene A. Weygandt. B. S. Manager, Technical Report Writing PhiIip L. Stetson, M. D., Ph. D. Associate Director, Analytical Chemistry Karen S. Regan, D. V. M., D. A. C. V. P., D. A. B. T. Director, Pathology and Veterinary Medicine BarbaraL. Smith, D. V. M., M. S., Ph. D., D. A. C. V. S. Director, Laboratory Animal Resources David Wilke, D. V. M., MS. Diplomate A. C. V. O. Consulting Ophthalmologist III. Studv Schedule:

Proposed Animal Receipt Date: May 15,2001 Proposed Experimental Stan Date: May 31,2001 Proposed Necropsy Date( s): August 30 3 1, 200 1 Proposed Experimental Termination Date: To be added by protocol amendment.

603 of 619 WIL 417002 Page 4 of 19 May 15.2001 To be added by protocol amendment. Proposed Draft Report Date:

IV. Test Article Data:

A. Identification: 1. 1,2 Trichloroethane (1, 1, ZTCE).

NH06019301 B. Lot Number:

C. Purity: 99.56% by weight (from Certificate of Analysis). To be confirmed by WIL Research Laboratories, inc.

D. Stability: To be demonstrated by WIL Research Laboratories, Inc.

To be documented by WIL Research Laboratories, Inc. E. Physical Description:

F. Storage Conditions:

G. Reserve Samples: Room temperature in a sealed container.

Retention samples will be collected and stored in accordance with Standard Operating Procedures.

H. Personnel Safety Data: Provided by the Sponsor, preferably in the fotm of a Material Safety Data Sheet.

At a minimum, technical staff will wear safety glasses. appropriate gloves, and facility clothing when handling or working with the test article. Additional personal protective equipment, such as a respirator, protective coveralls or sleeve covers, may be required. Specific equipment and its use will be defined by study management and communicated to technical staff, as appropriate.

V. Test Svstem:

A. Species: Rat and mouse Fischer 344 CDF@( F 344) Crl: BR rat B6C3Fl mouse, B6C3FlKrl: BR. B. Strain:

604of 619 wlL 417002 PageSOf May 15.2ool c. source: The facihty from which animals were received wil1 be documented in the study records.

D. Number on Study: Rats: Forty males and 76 females will be placed on study. Approximately 50 males and 86 females will be purchased. Mice: Thirty six females will be placed on study. Approximately 46 female mice will be purchased.

E. Approximate Age and Weight: Animals will be approximately 7 to 9 weeks of age at exposure initiation. Females will be nulliparous and nonpregnant.

Rats: The males will weigh approximately 150 220 grams and the females will weigh approximately 100 160 grams at exposure initiation. Individual weights will be within k 20% of the mean for each sex.

Mice: The female mice will weigh approximately 18 26 grams at exposure initiation. Individual weights will be within 50% of the mean.

F. Disease Screen: Approximately seven days after receipt of animais, blood will be collected from the vena cava (under isoflurane anesthesia) of five rats per sex and five female mice for serological screening for pathogens. These animals will then be euthanized (by exsanguination) and discarded. For rats, serum samples will be analyzed for antibodies to the following pathogens (testing laboratory documented in study records): Sendai, RCVKDA, Reo, KRV, LCM, H l, PVM, ROPV viruses, hf. pulmonis and CARf3. For mice, the 605of619 wIL417002 Page6of 19 May 15.2001 screening profile will include: Sendai, EDIM. GDVIl, MHV. PVM, MPV, MVM, and h4. pulmonis.

G. Identification System: Rats will be uniquely identified by a Monele metal eartag displaying the animal number. Mice will be uniquely identified by a tail tattoo. Individual cage cards will be affixed to each cage and will display the study number, group number exposure level. animal number, sex and the dates of animal arrival and initiation of exposure.

H. Justification for Selection: These species and strains are generally recognized to be appropriate for subchronic inhalation toxicity and pharmacokinetic studies and are recommended in the TSCA guideline.

VI. Specific (Nonexposure Period) Maintenance Schedule:

A.

B. Animal Housing:

The rats and mice will be housed in separate environmentally controlled rooms.

Rats will be housed individually in clean suspended wire mesh cages in an environmentally controlled room. Mice will be housed three per cage in clean suspended wire mesh cages in an environmentally controlled room for approximately five days following receipt. Thereafter, mice will be housed individually. The cages will be elevated above cage board or other suitable material which will be changed at least three times each week. The facilities at WK Research Laboratories. Inc. are fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AALAC International).

Environmental Conditions:

Controls will be set to maintain room temperature at 72' 2 4° F (22 + 3° C) and relative humidity at approximately 30 708. Ruorescent lighting controhed by light timers will provide illumination for a I2 hour IQ&/ dark photoperiod. The light/ dark cycle may be interrupted for protocol specified activities. Temperature and relative humidity will be recorded once daily.

:: Gk HX: iii: ,g. PI 2: .:::, ::: ;) i; /; jcj/: ~::: pi .::::: : :::::: i.__ ... _..:: i::.: i . `:: " :: ._/:.. .., ~ I: c :: ::: t f gg i:,: $ ii: i / :i m.

606 of 619 WIL. 417002 Rgc7cf19 Ma? 15. tool C. Drinking Water:

Reverse osmosis treated tap water will be available ad libirum (except during exposures for rats). Water will be available to exposure cage batteries holding mice during inhalation exposures. Filten servicing the automatic watering system will be changed reguularly according to WIL Standard Operating Procedures. Municipal water supplying the laboratory will be analyzed for contaminants according to Standard Operating Procedures to ascertain that none are present in concentrations that would be expected to affect the outcome of the study.

D. Basal Diet:

PMI Nutrition International, Inc. Certified Rodent LabDiet' 5002 will be offered ad libirum during the study (except during exposures). Each lot utilized will be identified and recorded. Analyses of the certified feed for the presence of heavy metals and pesticides wili be provided by the manufacturer to ensure that none are present in concentrations that would be expected to affect the outcome of the study. Feeders will be changed and sanitized once per week.

VII. Experimental Des&:

A. Animal Receipt and Acclimation:

Each animal will be inspected by a qualified technician upon receipt. Animals judged to be in good health and suitable as test subjects will be acclimated for at least 14 days. All animals will be sexed, weighed and assigned a permanent animal number. During acclimation, each animal will be observed twice daily for changes in general appearance and behavior. All animals will receive a detailed physical examination approximately one week prior to dosing and at the time of animal selection for randomization.

B. Randomization:

At the conclusion of the acclimation period. animals judged to be suitable for testing will be assigned to groups using stratified randomization based on body weight (toxicity study groups). Female rats and mice will be assigned to the pharmacokinetic study using simple randomization by weight. Healthy female rats not assigned to the toxicity study groups will make up the group assignment pool for the pharmacokinetic group. At this time, the animal numbers and corresponding body weights wiIi be entered into the WIL Toxicology Data Management System (WTDMSTM), and a printout containing the animal numbers and individual exposure group assignments will be generated. Animals will then be arranged into the groups according fo the printout. Three 607 of 619 WIL417cn2 Paec 8 of19 Mav IS. zcmt WTDMS" protocoIs will be used, including one for the toxicity study rats, one for the kinetic study rats and one for the kinetic study mice.

.C. Route and Rationale of Test Material Administration:

The route of administration will be whole body inhalation exposure since this is a potential route of human exposure.

D. Organization of Test Groups, Exposure Levels and Treatment Regimen:

1. Organization of Test Groups:

The following table presents the study group arrangement.

a Actual target exposure levels will be based on the results of an acute inhalation toxicity study (WIL 4 17001) and will be defined m a protocol amendment. b Groups 5 and 6 will be exposed using the same exposure system and methods used for Group 4. Data for the toxicokinetic groups will be collected in separate WTDMS* computer protocols (from the toxicology goups).

Filtered air or test article vapor will be administered as daily six hour, whole body exposures on a five day per week basis for thirteen consecutive weeks (minimum of 65 total exposures). On the day following the final exposure, 10 males and 10 females per group will be euthanized and evaluated (clinical and anatomical pathology). The first day of exposure is defined as exposure day zero.

The pharmacokinetic component of this study consists of exposure and blood collection and processing for 36 female F 344 rats and 36 female B6C3Fl mice exposed at the high concentration. Sample analysis methods, analytical records and data and interpretation of pharmacokinetic data are part of a separate study and protocol and are not included in this study. The 608of619 wtL 017002 Pape 9 of 19 Mav IS. 2cnl E. Exposure Methods:

F. exposure regimen for the pharmacokinetic groups is six hours/ day, five days/ week for four weeks. On the Is*, 3", and 5' days of the fourth week of exposure for these groups, blood will be collected from 3 animals per specie per time point a 4,6,6.25 and 8 hours after the start of exposure.

Exposures will be conducted in stainless steel and glass whole body exposure chambers. One chamber will be dedicated for each group for the duration of the study. For the high concentration groups, the toxicology and kinetic group rats and the kinetic group mice will be exposed in the same inhalation chamber. This is considered to be acceptable for 6 hour exposures and for the shon duration of the kinetic study (28 days). The chambers will be operated under dynamic conditions from a HEPA and charcoal filtered air source.

Air flow rate through the chamber will be such that there will be at least 10 air changes per hour. Daily average chamber temperature and relative humidity will be 13t" C and 30 70%, respectively. These parameters will be monitored continuously and recorded approximately every 35 minutes during each day's exposure. Oxygen content of the exposure atmosphere will be measured during the methods developmental phase of the study and will be at least 19%.

All animals will be caged individually during the exposures. Food and water will not be available during the exposures.

All animals will be housed in normal animal colony rooms during non exposure hours. For each days' exposure, the animals will be transferred to exposure caging, transported to the exposure chamber room, exposed for the requisite duration and returned to their home cages.

Test Atmosphere Generation Methods:

If appropriate for 1,1.2 trichloroethane, vapors will be generated using a glass bead column type vaporization system. typically operated as follows: Test article and air carrier gas are metered to the column with air entering below the glass beads while the test article is deposited on the beads. The beads may be heated using a heating tape wrapped around the coIumn. An electric controller and thermocouple (positioned inside the bead column) are used to provide an appropriate, stable temperature and avoid overheating. Vaporization occurs as the test article flows over the surface of the beads and air moves up through the beads. The concentrated vapors are piped to the chamber inlet where the concentration is reduced by mixing with chamber ventilation air. Tubing and/ or pipes between the vaporization column and the chamber inlet may also be heated to avoid condensation of the vapor.

609 of 619 t? ane toor 19 wrL. 417cQz Mav 15. tom Additional vaporization approaches, such as bubbling air through minimally heated test article or vaporization of a fine test article aerosol, are available and may be used, if required. Details of generation system methodologies will be defined during pre study method development. Therefore, this information will be added as a protocol amendment after preliminary methods evaluations are conducted.

G. Methods for Characterization of Exposure Atmospheres:

1. NominaI Concentrations:

Nominal exposure concentrations wiIl he calculated for all exposures.

2. Actual Exposure Concentrations:

Actual test article vapor concentrations in the exposure atmospheres wiI1 be determined at least at hourly intervals for each chamber using an appropriate gas chromatographic method. Details concerning specific methods to be used and the frequency of determinations will be defined during pre study method development. Therefore, this information will be added as a protocol amendment after preliminary methods evaluations are conducted.

3. Temporal Stability and Homogeneity of Vapor Test Atmospheres:

During pre study method development trials, the stability of the test article vapor concentrations over time and the spatial homogeneity of the test article vapor concentrations will be evaluated for each test article chamber. Methods and results of these evaluations will be documented in the study records and described in the study report.

4. Chemical Purity in Vapor Test Atmosphere:

During pre study method development trials, the chemical purity of the vapor in the high concentration chamber will be evaluated. In general, the gas chromatograph will be optimized in an attempt to allow integration and peak area count determination for all peaks resulting from the test article. For comparison to the expected 1,1.2 TCE retention time, a high concentration gas bag standard wiI1 be prepared using a I. l, Z TCE reference material or the test article and analyzed prior to chamber sampling. The chamber atmosphere will be sampled and analyzed and the ?k of total peak area counts represented by the area counts for the 1,1,2 TCE peak will be calculated. High concentration chamber sampling and analysis will be 610of 619 wIL 417002 Page 11 of19 May 15.2001 performed in triplicate. Additional details concerning specific methods to be used wiI1 be added by protocol amendment.

5. Verification of Absence of Aerosols in Test Atmosphere:

During pre study method development, the high concentration chamber will be evaluated during vapor generation for the presence of aerosols. This test will be performed using a light scattering type real time aerosol monitor that has been optimized for detection of low levels of aerosols.

H. Test Article Characterization:

Purity Analysis:

Samples of the test article (5 10 mL), coltected from the original storage container( s) prior to use for animal exposures, will be assessed for purity using GUFID analysis of at least duplicate injections of the undiluted sample. The test article purity wiIl be expressed as `area percent."

Stability Analysis:

The same procedure used for the purity analysis will be followed for the stability analysis of sample( s) collected after completion of animal exposures or after completion of use of test article from an individual container. The resulting "area percent" values will be compared to the original purity values.

VIII. Experimental Observations:

A. Viabiiity Observations (All animals):

A11 animals will be observed for mortality/ moribundity twice daily, once in the morning and once in the afternoon. Moribund animals will be euthanized to ensure that tissues wiII not be lost due to autolysis.

B. Clinical Observations (All animals):

Chnical observations will be conducted after the exposure on exposure days. Observations will include, but are not limited to, changes in the skin, fur, eyes and mucous membranes, respiratory, circulatory, autonomic and central nervous system functions, somatomotor activity and behavior patterns. Findings noted at the clinical examination will be recorded for individual animals; the condition of animals without signs will be documented as a general comment.

611 of 619 WlLJ1700? Pa! zc 12 of 19 Mav 15.2001 C. Detailed Physical Examinations (Toxicology Groups):

All animals will receive a detailed physical examination approximately one week prior to and on the day of group assignment, weekiy during the exposure period, and on the day following the final exposure (prior to necropsy). The animals will be removed from their home cages and placed in a standard arena for observation. Observations wiIl be detailed and carefully noted. When appropriate. explicitly defined scoring systems will be used if, in the opinion of the study director, doing so increases the utihty of the data. Signs noted shall include, but are not limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e. g., lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling, as well as the presence of clonic or tonic movements, stereotypes (e. g., excessive grooming, repetitive circling) or bizarre behavior (e. g., self mutilation, walking backwards) will be recorded. The absence or presence of findings will be recorded for individual animals. Animals to be euthanized prior to scheduled necropsy will undergo an additional detailed physical examination prior to euthanasia to assist in determination of the cause of death.

D. individual Body Weights (AU animals):

Individual body weights will be recorded weekly, beginning one week prior to test material administration. For the toxicology group animals. a fasted weight wit1 be determined prior to blood collection and necropsy.

E. Individual Food Consumption (Toxicology Groups):

Individual food consumption will be recorded weekly for all animals, beginning one week prior to test material administration. Measurement will be for 7 day interval.

F. Ophthalmological Examinations (Toxicology Groups):

Ocuiar examinations will be conducted on all animals by a board certified veterinary ophthalmologist, using an indirect ophthalmoscope and/ or a slit lamp or other equivaIent suitable equipment, preceded by pupillary dilation with an appropriate mydriatic agent. The examinations will be performed before exposure initiation and near exposure termination.

612 of 619 G. Clinical Pathology (Toxicology Groups):

The following hematologic and biochemical parameters will be evaluated on all surviving animals just prior to the scheduled necropsy. These animals wili be fasted overnight before taking blood samples. Blood samples will be collected via the vena cava from animals under deep anesthesia from isoflurane inhalation.

1. Hematology:

Hemoglobin Hematocrit Etyhroc yte count Total Leukocyte count Differential leukocyte count Activated partial thrombopfastin time 2. Blood Chemistry:

Alanine aminotransferase Aspartate aminotransferase Alkaline phosphatase Phosphorus Glucose Urea nitrogen Creatinine Total cholesterol Triglycerides Gamma glutamyl transpeptidase Platelet count Reticulocyte counts (if signs of anemia are present) MCH .MCV MCHC Prothrombin time Total bilirubin Total protein Albumin Globulin A/ G ratio Calcium Chloride Potassium Sodium Additional serum chemistry parameters may be necessary to provide an adequate toxicological evaluation of 1. 1 , 2 TCE. If required, specific parameters will be added by protocol amendment (may result in additional cost).

Ii. Anatomic Pathology:

1. Macroscopic Examination (Toxicology Groups):

A complete necropsy will be conducted on all animals dying spontaneously, euthanized in e. nrernis or at the scheduled necropsy. Necropsy will include examination of the external surface, all orifices and the cranial, thoracic, abdominat and pelvic cavities including viscera. At the time of necropsy the following tissues and organs will be coliected and placed in 10% neutral buffered formalin (except as noted):

613 of 619   F@ e I4Of 19 WILd17002 May IS. ZOO1 Adrenals (2) Aorta Bone with marrow (sternum) Bone marrow smear (taken from femur") Brain (forebrain, midbrain. hindbrain) Epididytnides (2) ' Eyes with optic nerve (2) b Exorbital lacrimal gland (2) Gastrointestinal tract Esophagus Stomach Duodenum Jejunum IIeum Cecum Colon Rectum Harderian glands (2) Heart Kidneys (2) Larynx Liver (sections of two lobes) Lungs (including bronchi. fixed by inflation with fixative at constant pressure) Lymph node (tracheobroichial and mediastinai) Mammary gland (females only) Nasal tissues' Ovaries with oviducts (2) Pancreas Parathyroids (if present) Peripheral nerve (sciatic) Pituitary Prostate Salivary glands [mandibular (2)] Seminal vesicles (2) Skeletal muscle (rectus femoris) Skin Spinal cord (3 sections  cervical. thoracic, lumbar) Spleen Testes (2) ' Thymus Thyroids Trachea Urinary bladder Uterus with vagina All gross lesions ' = b Not placed in formalin; to be examined only if scientifically warranted. = The eyes will be placed in Davidson's solution. ' = The enure head will be removed and preserved. Following decalcification. six CTOSS

sections of the nasal cavities will be prepared for microscopic exminatlon in accordance with the method described by Morgan'. d = Parathyroid glands will be examined rmcroscopicaily if in the plane of section and in all cases when a gross lesion IS noted. ' = The testes and eptdidymides will be placed in Bouin's solution.

2. Organs Weights:

The following organs weights will be obtained from a11 animals at the time of necropsy. The thyroids will be weighed following fixation. (Paired organs will be weighed together):

614 of 619 WlLJ17002 paec ISof 19 Ma\ IS. 1001 I. Adrenals BMI Epididymides Heart Kidneys Liver Lungs (prior to inflation with fixative) Ovaries Spleen Testes Thymus Thyroids (with parathyroids) a Organ to body weight ratios will be calculated. a= weighed after fixation 3. Microscopic Examination:

Microscopic examination of hematoxylin eosin stained paraffin sections will be performed on the tissues listed in protocol section WII. H. 1. for ah animals in the control and high level grouts and from all rats that die or are euthanized in exrremis. The larynx. lungs, trachea, nasal tissues, liver, kidneys and all gross lesions will be examined from all animals in the low and mid level groups. If a potential target organ is noted in the high level group, that tissue will be examined in the low and mid level groups. Special stains may be used at the discretion of the pathologist to further characterize lesions and changes. Any special stains used will be documented in the individual animal data and interpretation of results will be included in the final report.

Blood Coliection and Processing for Pharmacokinetic Groups:

On days I, 3 and 5 of the fourth week of exposure for the pharmacokinetic group rats and mice, blood will be collected from subgroups of 12 animals per specie on each day. The blood collection time points will be 4. 6, 6.25 and 8 hours following the exposure start and three animals per specie will be used for each time point.

To accommodate the Chour time point, technicians equipped with appropriate protective equipment, including Tyvek@ coveralls and supplied air respirator, will quickly open the high concentration chamber (during exposure), remove designated animals (3 rats and 3 mice) and then reseal the chamber. The same procedure will be used for the 6 hour time point, except the generator will be turned off at 6 hours. As quickly as possible, the animals scheduled for blood collection at a specific time point (and day) will be anesthetized using isoflurane and blood will be collected from the heart (open chest cardiac stick) or vena cava. The blood will be mixed with an anticoagulant and a specific volume will then be placed in a head space analysis vial. The vials will be placed &.

615 of 619 WILJi700?

Papc 16619 May 15.301 Additional information regarding storage and shipment or transfer of the samples will be added by protocol amendment.

Following blood collection, the anesthetized animals will be exsanguinated to complete euthanasia. Organs/ tissues required by the Sponsor will be collected and each organ/ tissue or a representative sample thereof will be frozen using liquid nitrogen. The organs/ tissues to be collected will be added by protocol amendment. Frozen tissues will be placed in an appropriate labeled vial or other container and stored at approximately 70° C. The Sponsor will determine if and when the tissues will be shipped, analyzed or discarded and these requirements will be addressed by protocol amendment.

IX. Termination:

Animals ordered for this study but not selected for use on this study will be transferred for use on other studies or euthanited with CO*.

Animals euthanized in exrremis or at scheduled necropsies wiIl be anesthetized with Isoflurane@ and exsanguinated.

X. Statkticai Methods:

All analyses will be two tailed with significance levels of 5% and 1%. Ali means will be presented with standard deviations. Analysis of weekly body weights, body weight changes and food consumption as well as clinica pathology values (excluding differential white cell counts other than segmented neutrophils and lymphocytes), and absolute and relative organ weights will be analyzed by a one way analysis of variance' and when statistical significance is noted, comparison of the control group to each treated group by Dunnett's test3.

XI. Oualitv Assurance:

The study will be audited by the WIL Quality Assurance Unit while in progress to assure comphance with Good Laboratory Practice reguiations, adherence to the protocol and to WlL Standard Operating Procedures. The raw data and draft final report will be audited by the WIL Quality Assurance Unit prior to submission to the Sponsor to assure that the final report accurately describes the conduct and the findings of the study.

This is a regulated study and will be included on the WI. L master list of regulated studies.

616 of 619 WIL. 41700? Ree 17of 19 Mav 15.2CKIl XII. Records to be Maintained:

All original raw data, as defined by WIL SOPS and the applicable GLPs, will be stored as described in protocol section XIKL in the Archives at WIL Research Laboratories, Inc.

XIII. Work Product:

Sponsor will have title to all documentation records, raw data, slides, specimens, or other work product generated during the performance of the study. All work product including raw paper data, mapetically encoded records and specimens will be retained at no charge for a period of ten years following issuance of the final report in the Archives at WT. L Research Laboratories, Inc. Thereafter, WIL Research Laboratories will charge an archiving fee for retention of all work product (at the yearly fee described in the study agreement). All work product will he stored in compliance with regulatory requirements.

XIV. Reports:

The final report will contain a summary, test material data. methods and procedures, appropriate individual animal and summary data tabIes, methods and results of test atmosphere generation and characterization. a copy of the protocol and amendments (if any) and an interpretation and discussion of the study results. The report will contain al1 information necessary to conform with current EPA TSCA specifications as listed in the 799.9346 guideline. The report will be comprehensive and shall attempt to detine toxicity and the no observed effect level( s) under the conditions of this investigation.

WIL Research Laboratories will provide one (1) copy of an Audited Draft Report (with an electronic copy of the text; the body of the report without tables), submitted in a timely manner upon completion of the study prior to issuance of the final report. One (1) revision will be permitted as part of the cost of the study, from which Sponsor's reasonable revisions and suggestions will be incorporated into the Final Report as appropriate. Additional changes or revisions may be made. at extra cost. it is expected that the Sponsor will review the draft repon and provide comments to WIL within a one (1) month time frame following submission. WIL will submit the Final Report within one (1) month following receipt of comments. One (1) unbound original and eight (8) copies of the Final Report will be provided; requests for additional copies of the Final Report may result in additional charges.

XV, Protocol Modification:

Modification of the protocol may be accomplished duting the course of this investigation. However, no changes will be made in the study design without the 617 of 619  WILJl7002 Rpe 18 of 19 May 1s. ICOI verbal or written permission of the Sponsor. In the event that the Sponsor verbally requests or approves changes in the protocol, such changes will be made by appropriate documentation in the form of protocol amendments. All alterations of the protocol and reasons for the modification( s) will be signed by the Study Director and Sponsor Representative.

XVI. Animal Welfare Act Compliance:

This study will comply with all applicable sections of the Final Rules of the Animal Welfare Act regulations (9 CFR). The Sponsor should make particular note of the following:

l The Sponsor signature on this protocol documents for the Study Director the Sponsors assurance that the study described in this protocol does not unnecessarily duplicate previous experiments.

l Whenever possible, procedures used in this study have been designed to avoid or minimize discomfort, distress or pain to animals. AI1 methods are described in this study protocol or in written laboratory standard operating procedures.

l Animals that experience severe or chronic pain or distress that cannot be relieved will be painlessly euthanized as deemed appropriate by the veterinary staff and Study Director.

l Methods of euthanasia used during this study are in conformance with the above referenced regulation.

XVII. References:

1. Approaches to the Identification and Recording of Nasal Lesions in Toxicology Studies, Morgan, Kevin, T., Toxicologic Pathology, Vol. 19, No. 4 (Pait I): 337 351 (1991).

2. Snedecor, G. W. and Cochran, W. G. (1980) One way classification: analysis of variance. In: Statistical Methods, Seventh Edition. Iowa State University Press, Ames, IA, pp. 215 337.

618 of 619 WIL 417002 Page 19of 19 May IS. 2001 3. Dunnett, C. W. (1964) New tables for multiple comparisons with a control, Biometrics, 20: 482491.

XVIII. Protocol Amroval:

HAP Task Force Sponsor approval received Sponsor Representative St; dy Director 619 of 619


[Federal Register Volume 84, Number 44 (Wednesday, March 6, 2019)]
[Rules and Regulations]
[Pages 8001-8003]
[FR Doc No: 2019-04069]

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ENVIRONMENTAL PROTECTION AGENCY

40 CFR Part 62

[EPA-R05-OAR-2018-0588; FRL-9990-45--Region 5]

Air Plan Approval; Minnesota; Commercial and Industrial Solid
Waste Incineration Units and Other Solid Waste Incineration Units
Negative Declarations for Designated Facilities and Pollutants

AGENCY: Environmental Protection Agency (EPA).

ACTION: Final rule.

-----------------------------------------------------------------------

SUMMARY: The Environmental Protection Agency (EPA) is notifying the
public that we have received negative declarations from Minnesota
pertaining to the presence of Commercial and Industrial Solid Waste
Incineration (CISWI) units and Other Solid Waste Incineration (OSWI)
units in Minnesota. The Minnesota Pollution Control Agency (MPCA)
submitted its CISWI negative declaration by letter dated February 3,
2017, and its OSWI negative declaration by letter dated June 21, 2017.
MPCA notified EPA in its negative declaration letters that there are no
CISWI or OSWI units subject to the requirements of the Clean Air Act
(Act) currently operating in Minnesota. On September 27, 2018, EPA
published a notice of proposed rulemaking for these declarations.

DATES: This final rule is effective on April 5, 2019.

ADDRESSES: EPA has established a docket for this action under Docket ID
No. EPA-R05-OAR-2018-0588. All documents in the docket are listed on
the http://www.regulations.gov website. Although listed in the index,
some information is not publicly available, i.e., Confidential Business
Information (CBI) or other information whose disclosure is restricted
by statute. Certain other material, such as copyrighted material, is
not placed on the internet and will be publicly available only in hard
copy form. Publicly available docket materials are

available either through www.regulations.gov or at the Environmental
Protection Agency, Region 5, Air and Radiation Division, 77 West
Jackson Boulevard, Chicago, Illinois 60604. This facility is open from
8:30 a.m. to 4:30 p.m., Monday through Friday, excluding Federal
holidays. We recommend that you telephone Margaret Sieffert,
Environmental Engineer, at<PHONE_NUMBER> before visiting the Region 5
office.

FOR FURTHER INFORMATION CONTACT: Margaret Sieffert, Environmental
Engineer, Environmental Protection Agency, Region 5, 77 West Jackson
Boulevard (AT-18J), Chicago, Illinois 60604,<PHONE_NUMBER>,
<EMAIL_ADDRESS>
SUPPLEMENTARY INFORMATION: Throughout this document whenever ``we,''
``us,'' or ``our'' is used, we mean EPA. This supplementary information
section is arranged as follows:

I. Background
II. What action is EPA taking?
III. Statutory and Executive Order Reviews

I. Background

    On February 3, 2017, MPCA submitted its CISWI negative declaration,
in which MPCA certified that there are no existing CISWI units
currently operating in Minnesota.
    On June 21, 2017, MPCA submitted its OSWI negative declaration, in
which it certified that there are no existing OSWI units currently
operating in Minnesota.
    On September 27, 2018, EPA published a notice of proposed
rulemaking (NPRM) proposing notice of Minnesota's negative declarations
for CISWI and OSWI. The specific details of Minnesota's request and the
rationale for EPA's approval are discussed in the NPRM and will not be
restated here. EPA did not receive any comments on the proposed action.

II. What action is EPA taking?

    EPA is notifying the public of EPA's receipt of MPCA's negative
declarations for both CISWI and OSWI facilities and amending 40 CFR
part 62 to reflect both negative declarations. For CISWI, EPA received
the negative declaration on February 3, 2017, and for OSWI, EPA
received the negative declaration on June 21, 2017. In this action, EPA
is finalizing its notification. EPA is also adding 40 CFR part 62.5880
and 62.5890 to reflect these notifications.

III. Statutory and Executive Order Reviews

A. General Requirements

    This action is not a ``significant regulatory action'' under the
terms of Executive Order 12866 (58 FR 51735, October 4, 1993) and
therefore is not subject to review by the Office of Management and
Budget under Executive Orders 12866 and 13563 (76 FR 3821, January 21,
2011). For this reason, this action is also not subject to Executive
Order 13211, ``Actions Concerning Regulations That Significantly Affect
Energy Supply, Distribution, or Use'' (66 FR 28355, May 22, 2001). This
action is not an Executive Order 13771 (82 FR 9339, February 2, 2017)
regulatory action because this action is not significant under E.O.
12866. This action merely approves state law as meeting Federal
requirements and merely notifies the public of EPA's receipt of
negative declarations from an air pollution control agency without any
existing CISWI or OSWI units in its state. This action imposes no
requirements beyond those imposed by the state. Accordingly, the
Administrator certifies that this rule will not have a significant
economic impact on a substantial number of small entities under the
Regulatory Flexibility Act (5 U.S.C. 601 et seq.). Because this rule
pertains to pre-existing requirements under state law and does not
impose any additional enforceable duty beyond that required by state
law, it does not contain any unfunded mandate or significantly or
uniquely affect small governments, as described in the Unfunded
Mandates Reform Act of 1995 (Pub. L. 104-4). This rule is not approved
to apply on any Indian reservation land or in any other area where EPA
or an Indian tribe has demonstrated that a tribe has jurisdiction. In
those areas of Indian country, the rule does not have tribal
implications and will not impose substantial direct costs on tribal
governments or preempt tribal law as specified by Executive Order 13175
(65 FR 67249, November 9, 2000). This action also does not have
Federalism implications because it does not have substantial direct
effects on the states, on the relationship between the national
government and the states, or on the distribution of power and
responsibilities among the various levels of government, as specified
in Executive Order 13132 (64 FR 43255, August 10, 1999). This action
merely provides notice of receipt of negative declarations, and does
not alter the relationship or the distribution of power and
responsibilities established in the Act. This rule also is not subject
to Executive Order 13045 (62 FR 19885, April 23, 1997), because it just
notifying the public regarding receipt of the negative declarations.
    In reviewing state plan submissions, EPA's role is to approve state
choices, provided that they meet the criteria of the Act. With regard
to negative declarations for designated facilities received by EPA from
states, EPA's role is to notify the public of the receipt of such
negative declarations and revise 40 CFR part 62 accordingly. In this
context, in the absence of a prior existing requirement for the state
to use voluntary consensus standards (VCS), EPA has no authority to
disapprove a state plan submission or negative declaration for failure
to use VCS. It would thus be inconsistent with applicable law for EPA,
when it reviews a state plan or negative declaration submission, to use
VCS in place of a state plan or negative declaration submission that
otherwise satisfies the provisions of the Act. Thus, the requirements
of section 12(d) of the National Technology Transfer and Advancement
Act of 1995 (15 U.S.C. 272 note) do not apply. This rule does not
impose an information collection burden under the provisions of the
Paperwork Reduction Act of 1995 (44 U.S.C. 3501 et seq.).
    The Congressional Review Act, 5 U.S.C. 801 et seq., as added by the
Small Business Regulatory Enforcement Fairness Act of 1996, generally
provides that before a rule may take effect, the agency promulgating
the rule must submit a rule report, which includes a copy of the rule,
to each House of the Congress and to the Comptroller General of the
United States. EPA will submit a report containing this action and
other required information to the U.S. Senate, the U.S. House of
Representatives, and the Comptroller General of the United States prior
to publication of the rule in the Federal Register. A major rule cannot
take effect until 60 days after it is published in the Federal
Register. This action is not a ``major rule'' as defined by 5 U.S.C.
804(2).
    Under section 307(b)(1) of the Act, petitions for judicial review
of this action must be filed in the United States Court of Appeals for
the appropriate circuit by May 6, 2019. Filing a petition for
reconsideration by the Administrator of this final rule does not affect
the finality of this action for the purposes of judicial review nor
does it extend the time within which a petition for judicial review may
be filed, and shall not postpone the effectiveness of such rule or
action. This action may not be challenged later in proceedings to

enforce its requirements. (See section 307(b)(2).)

List of Subjects in 40 CFR Part 62

    Administrative practice and procedure, Air pollution control,
Environmental protection, Commercial and Industrial Solid Waste
Incinerators, Intergovernmental relations, Other Solid Waste
Incinerator Units, Reporting and record-keeping requirements.

    Dated: February 25, 2019.
Cheryl L. Newton,
Acting Regional Administrator, Region 5.

    40 CFR part 62 is amended as follows:

PART 62--APPROVAL AND PROMULGATION OF STATE PLANS FOR DESIGNATED
FACILITIES AND POLLUTANTS

0
1. The authority citation for part 62 continues to read as follows:

    Authority:  42 U.S.C. 7401 et seq.

0
2. Add Sec. Sec.  62.5880 and 62.5890 and their undesignated center
headings to subpart Y to read as follows:
* * * * *

Control of Air Emissions From Commercial and Industrial Solid Waste
Incineration Units

Sec.  62.5880  Identification of plan--negative declaration.

    On February 3, 2017, the Minnesota Pollution Control Agency
submitted a negative declaration letter to EPA certifying that there
are no existing Commercial and Industrial Solid Waste Incineration
(CISWI) units in the State of Minnesota subject to the emissions
guidelines at 40 CFR part 60, subpart DDDD.

Control of Air Emissions From Other Solid Waste Incineration Units

Sec.  62.5890  Identification of plan--negative declaration.

    On June 21, 2017, the Minnesota Pollution Control Agency submitted
a negative declaration letter to EPA certifying that there are no
existing Other Solid Waste Incineration (OSWI) units in the State of
Minnesota subject to the emissions guidelines at 40 CFR part 60,
subpart FFFF.
[FR Doc. 2019-04069 Filed 3-5-19; 8:45 am]
 BILLING CODE 6560-50-P