311'1705 

P.O. Box 331, Millwood, VA 22646 (540) 837-1602 

August 7, 2012 

BY I-lAND 

Document Control Ortice 

Oftice of Pollution 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 of Ethylene 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 of the Enforceable Consent Agreement for Ethylene Dichloride, 

thc I lAP Task Forcc submits the enclosed addendum to the study plan for an extended onegeneration drinking \\'<.1ler reproductive toxicity study of ethylene dichloride in rats, tiled on 

July 2. 2012. The addendum is an amendment to Appendix C, the protocol for the extended 

one-generation drinking ''ater reproductiYe toxicity study of ethylene 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 if there are any questions. 

1111111111111111111111111111111111111111111111111111111111111111 

4 0 1 2 0 0 0 0 0 8 3 

Sincerely, 

Pe!:f~L~, ~J~f /vJ 0V 

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 Task Force 

An Extended One-Generation Drinking Water Reproductive Toxicity Study of Ethylene 

Dichloride in Rats 

Pmtocol Modifications: 

1) 4.7 Storage Conditions: 

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 of this section is 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 of Test Substance-Treated Drinking Water; 

The fourth sentence of this section is revised as follows: 

These samples will be analyzed on the day of collection 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 2 01 2 
Page 2 of 12 

5) 7.5.5 Reserve Samples: 

This section is removed from the protocol. 

6) 7.6 F0 Determination of Estrous Cycles: 

The last sentence of this section is revised as follows: 

WIL-417005 

Protocol Amendment 1 

An analysis of the female cycle pattern (e.g., regularly cycling, irregularly cycling, 

etc.) will be reported. 

7) 7.9 Standardization of F1 Litter Size and Identification: 

The second paragraph ofthis section is replaced with the 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 I 0% 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 of culled 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 stimulating hormone (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 I O<% 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 of Ft Animals to Cohorts 

The first sentence of paragraph 2 is clarified to indicate that pups will be assigned to 

one of 4 cohorts. Also, the table in this section is revised as follows: 

Cohort Number of Animals Primary Assessments 

lA 1 /sex/litter/group Primary reproductive/ developmental 

(>20/sex/group) toxicity testing 

lB 1 /sex/litter/group Follow-up reproductive assessment, if 

(2:20/sex/group) required 

2A 1 pup/litter (2:10/sex/group, Neurobehavioral testing 

representing_ 2:20 litters) 

2B I pup/litter (2: 1 0/sex/group, PND 21 Neuropathology 

representing 2:20 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 morpho logy and organs of the 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 of collection will be changed to 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 

l 0 randomly selected pups/sex/group (representing 20 litters/group, if possible) will 
Page 4 of 12 

WIL-417005 

Protocol Amendment 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 Parental Appearance and Behavior: 

The following is added to the second paragraph: 

All detailed physical examinations will be performed by an individual trained to 

perform the 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 second paragraph: 

All detailed physical examinations will be perfonned by an individual trained to 

perform the 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 of necropsy). 
Page 5 of 12 

18) 7.15.1 Auditory Startle Response: 

The following is clarified in the protocol: 

WIL-417005 

Protocol Amendment 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 ± 5 dBA. 

Also, the last sentence of this section is replaced with the 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 of primary 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 graphically presented. 

19) 7.15.2 Locomotor Activity: 

The following is clarified in the protocol: 

The white noise generation system will be set to operate at 70 ± 10 dBA. 

Also, the last sentence ofthis section is replaced with the 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 ± I 0 dBA. 

21) 7.16 Production of a Second Generation (F1 Cohort lB): 

The title of this section is changed as indicated above. 

22)7.17 Euthanasia of Ft Reproductive/Developmental Toxicity (Cohorts lA and 

lB) and Post-Weaning Moribund Animals: 

The title of this 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 collection period). 

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 of corpora 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 of tissues/organs to 

be collected: 
Adrenals (2) 

Aorta (1) 

Bone with marrow 

(sternebrae) 

Brain (cerebrum, brain stem, 

cerebellum) (3) 

Coagulating glands (2t 

Eyes with optic nerve (2)b 

Gastrointest ina) 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 (including bronchi, 

fixed by inflation with 

fixative) ( 5) 

Lymph nodes 

Mediastinal ( 1) 

Mesenteric ( 1) 

Mediastinal tissues ( 1) 

Mesenteric tissues ( 1) 

Nasal tissues/pharynx (2)c 

Oral tissues (2) 

Ovariesd,e and oviduct (2t 

Pancreas (1 t 

Peripheral nerve (tibial) 

Pituitary (1 t 

Prostate ( 1 t 

Mandibular salivary 

glands (1) 

Seminal vesicles (2f 

Skeletal muscle (rectus 

femoris)( 1) 

WIL-417005 

Protocol Amendment 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 with Tracheai 

[with parathyroids if 

present ( 1) t 

Urinary bladder 

Uterus with cervix and 

vagina (4t 

All gross lesions 

(all groups t 

The number in parentheses is the minimum number of sections to be examined if the 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 F 1 Cohort I B animals if Cohort lA results are equivocal or if reproductive or 

endocrine toxicity is suspected. 

b- Eyes with optic nerves will be fixed in Davidson's solution. 

c - Following fixation in formalin, nasal tissues will be decalcified and sections of 6 nasal levels will be 

prepared for microscopic examination as defined by SOP T4-016 (as revised). 

d Ovaries will be fixed in I 0% 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 I A females for quantitative evaluation of primordial follicles. 

f- For females; a corresponding section of skin 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 and right organs. 

h -If the 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 (if present) 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 replaced with the 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 

lie art 

Kidneys 

Liver 

Ovariesa 

Pituitary" 

A Also collected from F1 Cohort lB animals 

* These paired organs will be weighed separately. 

Prostate/\a 

Seminal vesicles with coagulating glands 

(with accessory fluids t 

Spleen 

Testes*a 

Thyroids with parathyroids+ 

Thymus 

Uterus (with oviducts and cervix)a 

" Tissue will be weighed after fixation in 10% neutral-buffered formalin 

'' If there 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 indicated tissues will be processed to the 

block stage (in paraffin) and will be examined (upon Sponsor approval, at additional 

cost) if results from Cohort 1 A are equivocal or in cases of suspected reproductive or 

endocrine toxicity. 

The first sentence of the second paragraph 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 ofthe third paragraph is revised as follows: 

Histopathological examination of the testis will include a qualitative assessment of 

the stages of spermatogenesis (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 

know ledge of group assignment. 

29) 8.4.1.2 Neuropathology- Microscopic Examination: 

The first sentence of this 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 

know ledge of group assignment. 

31) 8.4.2.2 Neuropathology - Microscopic Examination: 

The first paragraph of this section is replaced with 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 corrected to 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 and Industrial Health 1999, 15, 48-64. 

National Research Council. Guide for the Care and Use of Laborat01y 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 

Histopathological Evaluation of the Testis. Cache River Press, Clearwater, FL, 1990. 

Reasons for Protocol Modification: 

1) Clarification of storage conditions. 

2-3) Clarification of the source/type of drinking water to be used on study. 

4) To an add additional stability time point. 

5) To remove unnecessary storage of reserve samples of formulations. 

6) To clarify what will be evaluated in the female estrous cycle pattern. 

7) To include macroscopic examination of culled offspring. 

8) To remove/correct minor redundancies/inaccuracies in the pooling of blood samples 

and the method of euthanasia. 

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 of blood collection. 

12) To clarify which organs will be collected and weighed from each set of animals. 

13-16) To clarify that the detailed physical observations performed weekly will be 

conducted by an FOB-trained person and to remove duplicative observations. 

17) To coincide the days of estrous cycle evaluation with the day of scheduled 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 of the relevant 

exposure and to indicate the need for documentation of the site of collection of blood 

samples. 

24) Added clarification that corpora lutea will only be counted through lactation day 4. 

25) Corrected number of lobes of the liver to 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) Corrected typographical error; also, clarification and reference added. 

28) Clarified that brains will be measured without knowledge ofthe group assignment. 

29) Clarified that only brains from Cohoti 2B pups will be prepared on PND 21. 

30) Clarified that brains will be measured without knowledge ofthe group assignment. 

31) Clarified that initial examinations will be conducted on control and high-dose group 

animals only, and that only brains will be examined for Cohort 2B pups on PND 21. 

32) Corrected the age of neuropathological evaluation for the Cohort 2B animals. 

3 3) Rc f'crenccs added/revised. 
Page 12 of 12 

VvlL-417005 

Protocol Amendment 1 

Sponsor's approval was obtained via~----- on~ (l1.1Jd0 ~~­

ate 

WIL Research 

HAP Task Force 

Melissa J. Beck, PhD 

Study Director 

-~ .. ~ 7oJt_ ~~~