# 311'1705 P.O. Box 331, Millwood, VA 22646 (540) 837-1602



### August 7, 2012 BY 

### I-lAND Document Control Ortice Oftice ofPollution Prevention and Toxics Room 6428 Environmental Protection Agency 1200 Pennsylvania A

### venue, NW Washington. D.C. 20460

Dear Sirs: Re: Testing Consent Order for Ethylene Dichloride; Study Plan for Extended One-Generation Drinking Water Reproductive Toxicity Study ofEthylene Dichloride in Rats (Docket No. OPPT-2003-00 I

### 0)**~** 

# = ......., ::tJ"' c:: Cl:;o Gi -cp1

I -:J ;:,-) 0) -i[T1

N 

### w



### Pursuant to Section IX 

### ofthe Enforceable Consent Agreement for Ethylene Dichloride, thc IlAP Task Forcc submits the enclosed addendum to the study plan for an extended onegeneration drinking \\'<.1ler reproductive toxicity study ofethylene dichloride in rats, tiled on July 2. 2012. 

### The addendum is an amendment to Appendix C, 

### the protocol for the extended one-generation drinking ''aterreproductiYe toxicity study ofethylene dichloride in rats. The amendment rdlects minor changes that arc consistent with the approYed EPA protocol and OECD 

### Guideline 4-J.J. 

### Si\ et)pies uf

### the study plan addendum are enclosed. Please do nnt hesitate to contact me ifthere are any questions.

1111111111111111111111111111111111111111111111111111111111111111 4 0 1 2 0 0 0 0 0 8 3 

### Sincerely,

# Pe!:f~L~,**~J~f**/vJ0

# V Manager Enclosures 

## CONTAINS NO CB/ cc: J. 

### Schaeffer \\'.Calley Norman, Esq.

\XIIII):," I

## AU~ 

# 23 

# 2012**/t~**

### PROTOCOL AMENDMENT 1 Study Number: WIL-417005

Sponsor: HAP TaskForce

An Extended One-Generation Drinking Water Reproductive Toxicity Study ofEthylene Dichloride

### in Rats PmtocolModifications: 

### 1) 4.7 StorageConditions: This section is revised as follows: 

### ln a room-temperature flammables cabinet 

### m a climate-controlled atmosphere, 

### under a nitrogen blanket. 2) 6.3 Drinking \Vater: The second sentence ofthis sectionis revised as follows: Reverse 

### osmosis-treated tap water (with test substance added during the treatment period for the animals assignee! to Groups 2-4) will be available ad libitum to all animals and will be provided tl-om standard plastic water bottles (approximately 250 mL). 

### 3) 7.4.3 Vchide (Drinking Water): This section is revised as follows: Reverse osmosis-treated tap water. 

### 4) 7.5.3 Stability ofTestSubstance-Treated Drinking Water; The fourth sentence ofthis section is revised as follows: These samples will be analyzed on the day ofcollection and for stability after 8 and 15 days. The minor changes described in this amendment are in compliance with the approved EPA protocol and OECD guideline.**~1JB2** 06 l\ug 201 2Page 2 of 12 5) 

### 7.5.5 Reserve Samples: This section is removed from the protocol. 6) 

### 7.6 F0 

### Determination ofEstrous Cycles: The last sentence ofthis section is revised as follows: WIL-417005 Protocol Amendment 1

An analysis ofthe female cycle pattern 

### (e.g., regularly cycling, irregularly cycling, etc.) will be reported. 7) 

### 7.9 Standardization ofF1 

### Litter Size and Identification: The second paragraph ofthis section is replacedwiththe following: Following selection and blood/thyroid collection for PND 4 thyroid hormone analysis and possible histopathology (See Section 7.10), any remaining culled pups will be euthanized by an intraperitoneal injection of sodium pentobarbital, and examined macroscopically. Any gross lesions will be collected and placed in 

### I0% neutralbuffered formalin, and the carcasses will be discarded. 8) 

### 7.10 Thyroid Hormone Analysis (PND 4 Culled Pups): The first paragraph ofthis section is revised as follows: On PND 4, culled pups will be anesthetized by isoflurane inhalation and as much blood as possible will be collected via cardiac puncture and pooled by litter until a total of 15 samples/dose level (minimum of 1.5 mL each) has been obtained. 

### -Bteet=l may be pooled by litter to ensure a sufficient volume fur hormone analysis. To the extent possible, samples fi·om the first 15 litters at each dose level with sufficient numbers ofculled pups will be used. Additional litters may be used if there is an insufficient number of pups to obtain the desired blood volume. The following parameters will be evaluated: Thyroxine (T4; Cobas e411) Thyroid stimulatinghormone (TSH; Radioimmunoassay 

### [RIA)) Also, the third paragraph ofthis section is replaced with the following: Following blood collection, the animals will be euthanized by decapitation and examined macroscopically. The thyroid glands will be harvested for thyroid hormone analyses. The trachea, with the thyroid gland attached, will be removed, placed into cassettes labeled to match the pooled blood samples, and placed in IO<% neutral-Page 3 of 12 WIL-417005 Protocol Amendment 1 buffered formalin for possible future histopathological examination. 1 f no histopathologic examination is required, these tissues will be discarded after acceptance ofthe final repot1. 9) 7.11 Assignment ofFt Animals to Cohorts The first sentence ofparagraph 2 is clarified to indicate that pups will be assigned to one of4 cohorts. Also, the table inthis section is revised as follows:
Cohort Number ofAnimals PrimaryAssessments

lA 1/sex/litter/group Primaryreproductive/developmental (>20/sex/group) toxicitytesting lB 1/sex/litter/group Follow-up reproductive assessment, if (2:20/sex/group) required 2A 1pup/litter (2:10/sex/group, Neurobehavioral testing representing_2:20 litters) 2B I pup/litter (2:1 0/sex/group, PND 21 Neuropathology representing2:20 litters)
1

0)7.12.1 Macroscopic Examination: This section is replaced with the following: Nonselected F, pups will be weighed and euthanized by carbon dioxide inhalation on PND 21. Blood will be collected from a subset of weanlings (15/sex/group) as described in Section 7.12.2, and the cranial, thoracic, abdominal, and pelvic cavities of all animals will be examined. Emphasis will be placed on evaluation of developmental morphology and organs ofthe reproductive system. The animals will not be fasted prior to euthanasia. Mammary tissue and any gross lesions will also be collected from all pups and placed in 10% neutral-buffered formalin for possible future histopathologic examination. 11)7.12.2 Blood Collection and Thyroid Hormone Analysis: The route ofcollection will bechangedto vena cava. 12)7.12.3 Organ Weights: This section is replaced with the following: The following organs will be weighed and placed in 10% neutral-buffered formalin for possible future histopathologic examination. The brains, spleens, and thymuses of l0 randomly selected pups/sex/group (representing 20 litters/group, ifpossible) willPage 4 of 12 WIL-417005 ProtocolAmendment 1 be weighed, \Vhile 

### only the thyroids will be obtained from the 15 pups/sex/group used for blood collection. Organ-to-final-body-weight ratios will be calculated. Brain Spleen Thymus Thyroids+

+ From the same 15 pups/sex/group used for blood collection. Tissue will be weighed after fixation in 10% neutral-buffered formalin. 13)

### 7.14.1 ParentalAppearance and Behavior: The following is added to the secondparagraph: All detailed physical examinations will be performed by an individual trained to performthe functional observational battery (FOB) using the FOB scoring criteria (as described in SOP Tl-112). 14)

### 7.14.2 Detailed Clinical Observations: This section is removed fi"om the protocol. 

### 15)7.14.6.1 Appearance and Behavior: The following is added to the secondparagraph: All detailed physical examinations will be perfonned by an individual trained to performthe FOB using the FOB scoring criteria (as described in SOP T1-112). 16)

### 7.14.6.6 Pup Detailed Clinical Observations: This section is removed from the protocol. 17)

### 7.14.6.8 Evaluation for First Estrus and Determination of Estrous Cycles (F1 

### Cohort 1A): The second paragraph ofthis section is revised as follows: Daily vaginal lavages will also be performed for all female offspring assigned to Cohort 1 A to determine the stage of estrus for 2 weeks, from PND 78 through PND 91 (the day ofnecropsy).

### Page 5 of 12 18) 7.15.1 Auditory StartleResponse: The following is clarified in the protocol: WIL-417005 ProtocolAmendment 1

The white noise generation system will be set to operate at 70 ± 

### 10 dBA. The broadband background white noise will be set to 65 ± 

### 5 dBA. The startle stimulus will be set to 115 ± 

### 5dBA. Also, the last sentence ofthis section is replaced withthe following: As per guideline, the mean response amplitude (on each block of 10 trials) is the primary variable and will be statistically analyzed; the latency to the maximum amplitude is recorded, but is an ancillary variable (i.e., not ofprimary importance to the question, but used to qualitatively evaluate the internal consistency of the recording system) and therefore will not be statistically analyzed. Both variables, however, will be graphicallypresented. 19) 7.15.2 LocomotorActivity: The following is clarified in the protocol: The white noise generation systemwill be set to operate at 70 ± 

### 10 dBA. Also, the last sentence ofthis section is replaced withthe following: Locomotor activity will be evaluated as ambulatory activity counts obtained for the 60-minute test session. 20)7.15.3 Functional Observational Battery (FOB): The fo !lowing is clarified in the protocol: The white noise generation system will be set to operate at 70 ± 

### I0 dBA. 21)7.16 Production ofa Second Generation (F1 

### Cohort lB): The title ofthis section is changed as indicated above. 22)7.17 Euthanasia of Ft Reproductive/Developmental Toxicity (Cohorts lA and lB) and Post-WeaningMoribund Animals: The title ofthis section is changed as indicated above.Page 6 of 12 23)7.18 Clinical Pathology Evaluations (Fo and Ft Cohort lA): The third sentence is clarified as follows: WlL-417005 Protocol Amendment 1

Treated or control water will be available during this procedure (animals will be fasted during the collectionperiod). Also, the following is added to this section: The site of blood collection, including right or left, will be documented for each animal. 24)8.1 Macroscopic Examination (Fo, Ft Cohorts 1A and lB, and Unscheduled Deaths): The last sentence ofthe first paragraph is revised as follows: For F0 females that were mated, the number ofcorpora lutea (through lactation day 4 only) and former implantation sites will be recorded. 25)8.1 Macroscopic Examination (F0, F1 Cohorts 1A and lB, and Unscheduled Deaths): The following corrections/additions (bolded) arc made to the list oftissues/organs to be collected:Adrenals (2) Aorta (1) Bonewith marrow

(sternebrae) Brain (cerebrum, brainstem,

cerebellum) (3) Coagulating glands (2t Eyes with optic nerve (2)b Gastrointestina) tract

Esophagus Stomach Duodenum Jejunum Ileum Cecum Colon Rectum Hcm1 Kidneys (2) Lacrimal/Harderian

glands (2) Larynx (I) Page 7 of 12

Liver (sections ofleft, right

and median lobes)(3) Lungs (includingbronchi,

fixed by inflation with fixative) (5) Lymph nodes

Mediastinal (1) Mesenteric (1) Mediastinal tissues (1) Mesenteric tissues (1) Nasaltissues/pharynx (2)c Oraltissues (2) Ovariesd,e and oviduct (2t Pancreas (1

# t Peripheralnerve (tibial) Pituitary (1

# t Prostate (1

# t Mandibular salivary

glands (1) Seminal vesicles 

### (2f Skeletalmuscle (rectus femoris)(1) WIL-417005 ProtocolAmendment 1

Skin with mammary

gland and subcutis(1)r Spinal cord (cervical,

thoracic, lumbar) (3) Spleen (I) Testes with epididymides aoh and vas deferens (1) '"' Tongue (0) Thymus Thyroids withTracheai

[with parathyroids if present (1)

# t Urinarybladder Uterus withcervix and

vagina 

### (4t All gross lesions

(all groups

# t

The number in parentheses is theminimum number ofsections to be examined ifthe tissue is chosen for histopathologic examination. All tissues will be collected from animals that are found dead or euthanized in extremis. a - May also be examined for F1 Cohort IB animals ifCohort lAresults are equivocal or ifreproductive or endocrinetoxicity is suspected. b- Eyes with optic nerves will be fixed in Davidson's solution. c - Followingfixation in formalin, nasal tissues will be decalcified and sections of6 nasal levels will be prepared for microscopic examination as defined bySOPT4-016 (as revised). d Ovaries will be fixed in I0% neutral-buffered formalin for approximately 48 hours following which all ovaries will be transferred to 70% Ethanol. e - An additional section will be taken from F0 females for qualitative evaluation; additional sections (See Section R.3) will be taken fi·om F1 Cohort IA females for quantitative evaluation ofprimordial follicles. f- For females; a corresponding section ofskin will be taken from the same anatomic area for males. g- Testis and epididymis (right only) will be fixed in modified Davidson's solution. Both testes and epididymides from males found dead or euthanized in extremis will be fixed in modified Davidson's solution. Care will be taken to ensure separation between the left andright organs. h -Ifthe left testis or epididymis is noted with a gross lesion, the left testis and epididymis will be fixed in modified Davidson·s solution and the right testis and epididymis will be homogenized according to section 7.20.3. i- Thyroids and parathyroids (ifpresent) will be removed from the trachea following fixation to ensure tissue structure is maintained during prosection.

### Page 8 of 12 WIL-417005 

### Protocol Amendment 1 

### 26)8.2 Organ Weights: This section is replacedwiththe following: The following organs will be weighed from 

### Fo animals and 

### Ft animals assigned to Cohort 1 A at the scheduled termination. Selected organs will also be weighed from F1 

### animals assigned to Cohort lB at the scheduled termination, as noted below. Organ-to-final-body-weight ratios will be calculated.

Adrenals Brain Epididymides* (total and caudat lieart Kidneys Liver Ovariesa Pituitary" AAlso collected from F1Cohort lB animals 

### * These paired organs will be weighed separately. 

### Prostate/\a Seminalvesicles with coagulating glands

(withaccessory fluids

# t Spleen Testes*a Thyroids withparathyroids+ Thymus Uterus (with oviducts and cervix)a

" Tissue will be weighed after fixation in 10% neutral-buffered formalin '' Ifthere is a treatment-related effect on prostate weights, the dorsolateral and ventral segments will

be dissected (after fixation) and weighed separately. 27) 8.3 

### Microscopic Examination 

### (Fo and 

### F1 Cohorts 

### lA and 1B): The last sentence in the first paragraph is revised as follows: For F1 

### animals assigned to Cohort lB, these indicatedtissues will be processed to the block stage (in paraffin) and will be examined (upon Sponsor approval, at additional cost) ifresults from Cohort 

### 1 A are 

### equivocal orin cases ofsuspected reproductive or endocrine toxicity. The first sentence ofthe secondparagraph is revised as follows: Tissues and organs fixed in 10% neutral-buffered formalin, or other fixatives (as noted in Section 8.1) 

### from animals scheduled for microscopic examination (as 

### noted above) will be embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) for microscopic evaluation. The fourth sentence ofthethird paragraph is revised as follows: Histopathological examination ofthe testis will include a qualitative assessment of the stages ofspermatogenesis 

### (Russell et al., 1990).Page 9 of 12 28)

### 8.4.1.1 Neuropathology- Macroscopic Examination: WIL-417005 Protocol Amendment 1

To clarify the protocol, the brain lengths and widths will be recorded without knowledge ofgroup assignment. 29)

### 8.4.1.2 Neuropathology- Microscopic Examination: The first sentence ofthis section is revised as follows: 

### Brains from F1 pups assigned to Cohort 2B will be prepared for microscopic neuropathologic examination as described in Section 8.4.2.2 30)

### 8.4.2.1 Neuropathology- Macroscopic Examination: To clarify the protocol, the brain lengths and widths will be recorded without knowledge ofgroup assignment. 31)

### 8.4.2.2 Neuropathology - Microscopic Examination: The first paragraph ofthis section is replacedwith the following: The tissues listed below (brains only from PND 21 pups) from pups perfused for macroscopic evaluation on PND 21 (Cohort 2B) and 78 (Cohot1 2A) will be prepared for microscopic neuropathologic examination. Brains from all groups will be processed and embedded; brains from the control and high-dose groups will be initially evaluated. 32)

### 8.4.3 Subjective Diagnosis: The age for the Cohort 2B animals is correctedto PND 21. 33) 

### 17 References: The following items are added/revised in this section: Gray Jr., L.E., Ostby, J., Monosson, E., Kelce, W.R. Environmental antiandrogens: low doses of the fungicide vinclozolin alter sexual differentiation of the male rat. 

### Toxicology andIndustrial Health 1999, 

### 15, 48-64. National Research Council. 

### Guidefor the Care and Use ofLaborat01y Animals. Institute of Laboratory Animal Resources, Commission on Life Sciences; National Academy Press: Washington, DC. 1996.Page 10 of 12 WIL-417005 Protocol Amendment 1

Russell, L.D., Ettlin, R.A., SinhaHikim, A.P., Clegg, E.D. Histological and HistopathologicalEvaluation ofthe Testis. Cache River Press, Clearwater, FL, 1990. Reasons for ProtocolModification: 1) Clarification ofstorage conditions. 2-3) Clarification ofthe source/type ofdrinking waterto be used on study. 4) To an add additional stabilitytime point. 5) To remove unnecessary storage ofreserve samples offormulations. 6) To clarify what will be evaluated in the female estrous cyclepattern. 7) To include macroscopic examination ofculled offspring. 8) To remove/correct minor redundancies/inaccuracies in the pooling ofblood samples and the method ofeuthanasia. 9) To clarify the protocol and correct the day of neuropathology for Cohort 2B IS corrected to PND 21. 

### 1

0)To correct the method of euthanasia and clarify that mammary tissue should be collected from all pups. 11)To update the site ofblood collection. 12)To clarify which organs will be collected and weighed from each set ofanimals. 13-16) To clarify that the detailed physical observations performed weekly will be conducted by an FOB-trainedpersonand to remove duplicative observations. 17)To coincide the days ofestrous cycle evaluation with the day ofscheduled euthanasia. 18-20) To correct a typographical error, and clarify for auditory startle response and locomotor activity assessments which data will be reported and statistically analyzed. 21-22) Correction of section titles to clarify which animals arc assigned to the relevant activities.Page 11 of 12 WIL-417005 Protocol Amendment 1 23)To clarify that the water that will be available to the animals will be ofthe relevant exposure and to indicate the need for documentation ofthe site ofcollection ofblood samples. 24)Added clarification that corpora lutea will onlybe counted through lactation day4. 25) Correctednumber oflobes ofthe liverto be used for tissue collection, added footnote (c) for nasal tissue/pharynx collection, and adjusted subsequent footnote lettering. 26)Clarified which organs will be collected from each Coh01i. 27)Correctedtypographical error; also, clarification and reference added. 28)Clarified that brains will be measured withoutknowledge ofthe group assignment. 29) Clarified that only brains from Cohoti 2B pups will bepreparedonPND 21. 30)Clarified that brains will be measured withoutknowledge ofthe group assignment. 31)Clarified that initial examinations will be conducted on control and high-dose group animals only, and that onlybrains will be examinedfor Cohort 2B pups on PND 21. 32)Corrected the age ofneuropathological evaluationfor the Cohort 2B animals. 33) Rcf'crenccs added/revised.

### Page 12 of 12 

### VvlL-417005 Protocol Amendment 1

Sponsor's approval was obtained

### via~-----**on~**

# (l1.1Jd0**~~­** ate WIL Research



### HAPTask Force MelissaJ. Beck, PhD 

### StudyDirector

# -~ 

# ..**~** 

# 7oJt_**~~~**