Oral repeated-dose systemic and reproductive toxicity of 6:2 fluorotelomer alcohol in mice

Accepted Manuscript Title: Oral Repeated-Dose Systemic and Reproductive Toxicity of 6:2 Fluorotelomer Alcohol in Mice Author: Pushkor Mukerji Jessica Caverly Rae Robert C. Buck John C. O’Connor PII: DOI: Reference:

S2214-7500(14)00156-5 http://dx.doi.org/doi:10.1016/j.toxrep.2014.12.002 TOXREP 144

To appear in: Received date: Revised date: Accepted date:

29-10-2014 1-12-2014 1-12-2014

Please cite this article as: P. Mukerji, J.C. Rae, R.C. Buck, J.C. O’Connor, Oral RepeatedDose Systemic and Reproductive Toxicity of 6:2 Fluorotelomer Alcohol in Mice, Toxicol. Rep. (2014), http://dx.doi.org/10.1016/j.toxrep.2014.12.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Oral Repeated-Dose Systemic and Reproductive Toxicity of 6:2 Fluorotelomer

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Alcohol in Mice

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Pushkor Mukerji1,2, Jessica Caverly Rae1, Robert C. Buck3, and John C. O’Connor1

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E. I. duPont de Nemours and Company, Inc., Haskell Global Centers for Health & Environmental Sciences, P.O. Box 30, Newark, Delaware, 19714

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Corresponding author

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E. I. duPont de Nemours and Company, Inc., Chemicals and Fluoroproducts, Wilmington, Delaware, 19805

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Running Title: Oral Repeated-Dose Systemic and Reproductive Toxicity of 6:2 Fluorotelomer

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Alcohol in Mice

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Send correspondence to:

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Pushkor Mukerji

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Haskell Global Centers for Health & Environmental Sciences

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P.O. Box 30

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Newark, DE 19714

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(302) 366-5341

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Fax (302) 366-5211

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Email: [email protected] Page 1 of 39

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ABSTRACT

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6:2 fluorotelomer alcohol (6:2 FTOH) was evaluated for potential systemic repeated-dose

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and reproductive toxicity in mice. 6:2 FTOH was administered by oral gavage to CD-1 mice as a

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suspension in 0.5% aqueous methylcellulose with 0.1% Tween-80 at dosages of 1, 5, 25, or 100

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mg/kg/day. The no-observed-adverse-effect level (NOAEL) for systemic toxicity was 25

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mg/kg/day (males) and 5 mg/kg/day (females), based on effects at higher doses on mortality,

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clinical observations, body weight, nutritional parameters, hematology (red and white blood

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cell), clinical chemistry (liver-related), liver weights, and histopathology (liver, teeth,

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reproductive tract, and mammary gland). However, 6:2 FTOH was not a selective reproductive

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toxicant. The NOAEL for reproductive toxicity was >100 mg/kg/day; no effects on reproductive

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outcome were observed at any dosage. The NOAEL for viability and growth of the offspring

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was 25 mg/kg/day, based on clinical signs of delayed maturation in pups, and reductions in pup

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survival and pup body weight during lactation at 100 mg/kg/day. While the severity of the

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effects was generally greater in mice than previously reported in CD rats, the overall NOAELs

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were identical in both species, 5 mg/kg/day for systemic toxicity and 25 mg/kg/day for offspring

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viability/growth. 6:2 FTOH was not a selective reproductive toxicant in either species; no

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effects on reproductive outcome occurred at any dose level, and any effects observed in offspring

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occurred at dose levels that induced mortality and severe toxicity in maternal animals.

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Keywords: 6:2 fluorotelomer alcohol; reproductive toxicity; mice

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1.0

INTRODUCTION Fluorotelomer alcohols [FTOHs; F(CF2CF2)nCH2CH2OH, where n > 3] are industrial raw

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materials used to manufacture fluorotelomer-based products. The performance of these products

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is largely derived from the fluorotelomer functionality, which is covalently bound as a side-chain

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to a polymer backbone, or to additional hydrocarbon groups to create fluorosurfactants (Rao and

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Baker, 1994; Taylor, 1999; Kissa, 2001; Buck et al., 2011a,b). Polymeric products provide oil

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and water repellency and stain release protection that cannot be achieved with non-fluorinated

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alternatives. Fluorosurfactants provide unparalleled surface tension lowering, wetting and

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leveling properties. Recently, major global fluorotelomer manufacturers have committed to

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replace historic long-chain products that are potential precursors to long-chain

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perfluorocarboxylic acids (PFCAs, CnF2n+1COOH, where n>7), such as perfluorooctanoate

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(PFOA) (Ritter, 2010; U.S. EPA 2014). Alternatives, which cannot break down to long-chain

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PFCAs, such as products based on short-chain 6:2 FTOH (C6F13CH2CH2OH, CAS# 647-42-7,

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6:2 FTOH) as a key raw material, have been approved by regulators .

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A substantial body of data describes 6:2 FTOH hazards, answering the call for more

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published information about alternatives (Wang et al., 2013). The developmental and

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reproductive toxicity of 6:2 FTOH in CD rats as well as the acute, genetic, and subchronic

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toxicity in CD rats have been reported, including a benchmark dose of 5 mg/kg/day (Serex et al.,

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2014; O’Connor et al., 2014). There was no portal of entry-specific toxicity observed for 6:2

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FTOH when comparing repeated-dose oral and inhalation studies (Serex et al., 2012; Serex et al.,

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2014). In vitro 6:2 FTOH metabolism in mouse, rat and human hepatocyctes and in vivo oral and

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inhalation single and repeated-dose metabolism and kinetics studies have been reported showing

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rapid metabolism and elimination (Gannon et al., 2012; Himmelstein et al., 2012; Russell et al.,

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2013). Finally, the toxicity of two principal metabolism and environmental degradation Page 3 of 39

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products, perfluorohexanoic acid, PFHxA (Loveless et al., 2009; Chengelis et al., 2009; Klaunig

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et al., 2014) and 5:3 acid, C5F11CH2CH2COOH (MacKenzie et al., 2013), have been reported. The primary objective of this study was to evaluate the systemic oral repeated-dose and

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reproductive toxicity of 6:2 FTOH in CD-1 mice. The CD-1 strain was chosen for consistency

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with previously published toxicological evaluations of fluorotelomer alcohols and metabolites in

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mice (Mylchreest et al., 2005a; Loveless et al., 2006; Henderson and Smith, 2007; Das et al.,

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2008; Loveless et al., 2008). The present evaluation of 6:2 FTOH in CD-1 mice was intended to

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provide a basis for comparison with previously published repeated-dose and reproductive effects

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of 6:2 FTOH in CD rats. Further, this evaluation encompasses the toxicological action and

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effects of the parent chemical and metabolites from its biotransformation, including PFHxA and

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5:3 acid, resulting from 6:2 FTOH exposure in mice.

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2.0

MATERIALS AND METHODS

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2.1 Test substance and administration The test substance, 1-octanol, 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluoro-, also known as 6:2

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fluorotelomer alcohol (6:2 FTOH; 99.7% purity; C6F13CH2CH2OH; CAS #647-42-7), was

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supplied by DuPont Chemicals and Fluoroproducts, Wilmington, Delaware, as a clear liquid. 6:2

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FTOH was formulated in a vehicle of 0.1% Tween-80 in 0.5% aqueous methylcellulose.

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Formulations were administered by intragastric intubation to achieve dosage levels of 0, 1, 5, 25,

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and 100 mg/kg body weight/day (mg/kg/day). Control animals were administered the vehicle

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alone. All animals were dosed once each day at approximately the same time (± 2 hours) at a

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dose volume of 5 mL/kg body weight (calculated from the most recently collected body weight).

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Analyses at five time points, including the beginning and end of the study, confirmed that

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the formulations for the 5, 25, and 100 mg/kg/day groups were homogeneous mixed at the

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targeted concentrations (± 15% nominal) and stable until use. The concentration and stability of

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the formulation for the 1 mg/kg/day group could not be verified for the first six weeks of the

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premating period for P1 males only, after which the formulation method was refined, such that

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concentration, homogeneity, and stability were consistently within the targeted range.

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2.2 Test species and animal husbandry Male and female Crl:CD1(ICR) mice were acquired from Charles River Laboratories,

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Inc. (Kingston, NY) and maintained in an AAALAC-accredited facility in accordance with the

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principles described in the Guide to Care and Use of Laboratory Animals (National Research

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Council, 1996). Animals were housed individually in solid bottom caging with bedding and

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and litters were housed with their dams during the lactation period. Throughout the study,

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animals were fed PMI® Nutrition International, LLC (St. Louis, MO) Certified Rodent LabDiet®

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5002 and provided with tap water ad libitum. Animal rooms were maintained on a 12-hour

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light/dark cycle (fluorescent light), a temperature of 22  4C, and a relatively humidity of 50%

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 20%.

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Males were approximately 39 days old at arrival and 50 days old at study start. Females (nulliparous and not siblings of the males) were approximately 57 days old upon arrival and 75

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days old at study start. Mice were released from quarantine for study use after an acclimation

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period of approximately 1 week based on adequate body weight gain and freedom from clinical

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signs of disease or injury.

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All mice were weighed approximately weekly and cage-site examinations were performed at least twice daily to detect moribund or dead animals. All mice were also examined

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approximately 1-3 hours after dosing for clinical signs of overt toxicity.

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The study was performed in compliance with GLP standards and in accordance with the

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OECD, Section 4 (Part 415) and U.S. EPA, OPPTS 870.3550 guidelines. Animals were divided

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by computerized, stratified randomization into five groups of 15 males and 15 females such that

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all animals were within 20% of the mean for the sex at study start. Parental (P1) male mice were

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dosed for approximately 70 days prior to mating, in order to encompass the entire spermatogenic

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cycle; and throughout the cohabitation period (< 2 weeks), up until the day before scheduled

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euthanasia. P1 female mice were dosed for approximately 14 days prior to mating, and

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throughout the cohabitation period (< 2 weeks), gestation, and lactation, up until the day before

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scheduled euthanasia. F1 males and females that were selected for developmental landmarks

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were dosed from post-natal day (PND) 21 until the day before scheduled euthanasia.

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Clinical observations, body weights, and food consumption data were collected on all P1 generation mice approximately weekly throughout the premating period. During the

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cohabitation period, one male and one female mouse within each dose group were housed

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continually in the male’s cage until successful mating was verified by an intravaginal copulation

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plug, at which time they were returned to individual housing. The day copulation was confirmed

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was designated as GD 0. For presumed pregnant females, clinical observations, body weight,

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and food consumption data were collected on GD 0, 4, 7, 11, 14, and 17. The day a litter was

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delivered was designated as lactation day (LD) 0. For dams that delivered a litter, clinical

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observations and body weights were collected on LD 0, 7, 14, and 21, while food consumption

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data were collected on LD 0, 7, and 14. The number of live and dead pups and clinical

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observations for the pups were recorded once daily during the postpartum period, and pup body

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weights were collected on LD 0, 4, 7, 14, and 21. On LD 4, litters were randomly reduced to

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eight pups each (four per sex when possible).

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For the 1, 5, and 25 mg/kg/day groups, offspring in the F1 litters of each treatment level

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were randomly selected (one animal/sex/litter when possible) to be evaluated for collection of

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developmental landmark data. For the 100 mg/kg/day group, all offspring were euthanized as

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weanlings (PND 21) due to concerns about their ability to survive without the dam, based on low

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pup body weights and high pup mortality. Clinical observations, body weights, and food

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consumption data were collected on all surviving F1 generation mice weekly from PND 21 until

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PND 40-43. The age and body weight at either vaginal opening or preputial separation were

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recorded.

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2.4 Anatomic pathology All P1 mice and F1 adults were euthanized by isoflurane anesthesia followed by

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exsanguination, after completion of the cohabitation period for P1 males (on test day 107-109),

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on postpartum day 21 for P1 females, on test day 59 for P1 females that did not deliver a litter,

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and after developmental landmark achievement for all F1 adults (on PND 40-43). F1 weanlings

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designated for gross evaluations were euthanized by isoflurane anesthesia followed by CO2

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inhalation. The order of euthanasia was stratified across groups.

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All P1 mice, F1 weanlings, and F1 adults underwent an examination of the external

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surface, all orifices, and the cranial, thoracic, abdominal, and pelvic cavities, including viscera.

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The following tissues from P1 mice were weighed and retained in an appropriate fixative: brain,

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liver (with gallbladder), and kidneys from males and females; testes and epididymides from

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males; and ovaries (with oviducts) and uterus (with cervix) from females. Relative organ

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weights (% of terminal body weight) were calculated. In addition, the following tissues from P1

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mice were retained in a fixative: nose (I & II) with teeth, pancreas; seminal vesicles, coagulating

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gland, and prostate from males; and vagina and mammary gland from females. F1 pups that

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either died or were euthanized prior to the scheduled necropsy were examined grossly.

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Microscopic examination of hematoxylin and eosin (H&E) stained paraffin sections were

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performed by a veterinary pathologist on all tissues collected from P1 animals from the control

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and high-dose groups, from all P1 pairs that failed to produce a litter, and from all parental

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animals dying spontaneously (i.e., found dead) or euthanized prior to scheduled necropsy. In

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addition, the following organs from all P1 adult mice from all dose levels were processed to

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slides and examined microscopically: liver, nose/teeth, ovaries, uterus, vagina, and mammary

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gland.

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2.5 Clinical Pathology Each group of P1 males and females was divided into two approximately equally sized subsets for hematology or clinical chemistry. Blood samples for hematology (5-8

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animals/sex/group) or clinical chemistry (6-8 animals/sex/group) measurements were collected

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from the vena cava while each animal was under isoflurane anesthesia.

The following parameters were assessed with a Bayer® Advia 120 hematology analyzer:

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red blood cell count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular (cell)

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volume (MCV), mean corpuscular (cell) hemoglobin, mean corpuscular (cell) hemoglobin

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concentration (MCH), red cell distribution width (RDW), absolute reticulocyte count (ARET),

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platelet count, white blood cell count, and differential white blood cell count. The following

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parameters were assessed with an Olympus® AU640 clinical chemistry analyzer: aspartate

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aminotransferase (AST), alanine aminotransferase (ALT), sorbitol dehydrogenase (SDH),

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alkaline phosphatase (ALKP), total bilirubin (BILI), urea nitrogen (BUN), creatinine (CREA),

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cholesterol (CHOL), triglycerides, glucose, total protein, albumin, globulin, calcium, inorganic

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phosphorus, sodium, potassium, chloride, and total bile acids (TBA).

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2.6 Statistical Analyses

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Litter survival and indices for mating, fertility, and gestation were compared using

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Fisher’s Exact test (Fisher, 1985) with a Bonferroni-Holm correction (Holm, 1979). For all other

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continuous numerical data, transformations were applied when appropriate, based on preliminary

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tests for homogeneity of variance (Levene, 1960) and normality (Shapiro and Wilk, 1965); and a

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one-way analysis of variance (Snedecor and Cochran, 1967) followed by Dunnett’s test

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(Dunnett, 1964) were conducted. For all statistical analyses, significance was judged at p < 0.05.

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3.0

RESULTS

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3.1 Mortality and clinical observations in P1 mice 6:2 FTOH-related clinical abnormalities and mortality were observed in P1 males and

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females at 100 mg/kg/day (data not shown). One male and two females at this dose level were

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found dead or humanely euthanized during the premating or gestation periods. Clinical signs of

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toxicity that preceded death in these animals included clonic and tonic convulsions, ataxia,

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tremors (head/upper body/forelimbs), hyperreactivity, increased muscle tone, lethargy, pallor,

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labored breathing, and gasping. Similar signs of toxicity were also observed in 3 additional mice

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(1 male and 2 females) in the 100 mg/kg/day dose group that survived to the scheduled

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euthanasia. When the clinical abnormalities did not result in death, they abated within

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approximately one week after the first occurrence. There were no 6:2 FTOH-related clinical

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observations or mortality in P1 males or females at dose levels ≤25 mg/kg/day.

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3.2 Body weight and nutritional parameters in P1 mice

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Among P1 males administered 100 mg/kg/day, mean values for final body weight (test

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day 105), body weight gain (test days 0-105), food consumption (test day 0-70), and food

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efficiency (test day 0-70) were 4.8%, 11.6%, 7.5%, and 12.6% lower than control means (not

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statistically significant), respectively, and were attributed to 6:2 FTOH (Table 1). There were no

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effects on body weight or nutritional parameters in P1 males at dose levels ≤25 mg/kg/day.

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For P1 females during premating and gestation, there were no effects on body weight or

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nutritional parameters at any dose level (Table 1). During the lactation period, P1 females

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administered 100 mg/kg/day exhibited statistically significantly lower body weight gain, food

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consumption, and food efficiency during the intervals LD 0-7 and LD 7-14, compared with Page 10 of 39

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controls, resulting in statistically significantly lower body weight on LD 7 and LD 14 (Table 1,

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Figure 1). Compared with controls, mean body weights were 20% lower on LD 14 and mean

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food consumption was 51% lower from LD 7-14. There were no effects on body weight or

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nutritional parameters in P1 females at dose levels ≤25 mg/kg/day.

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3.3 Reproductive performance of P1 mice

There were no effects in reproductive performance (mating index, gestation index,

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fertility index, precoital interval, gestation length, number of implantation sites, or post

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implantation loss) at any dose level (Table 2).

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3.4 In-life data for F1 offspring

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Litters of dams administered 100 mg/kg/day exhibited 6:2 FTOH-related reductions in pup survival and body weight (Figure 2), and clinical abnormalities associated with delayed

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maturation (data not shown). At 100 mg/kg/day, pup survival was statistically significantly

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lower than control on LD 14 and 21, and over the interval LD 4-21 (45% reduction in lactation

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index; Table 2). Mean pup body weights were statistically significantly lower than control on

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LD 4, 7, 14, and 21, achieving a decrement of 65% lower than control on LD 21. Pups in four

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litters at this dose level were observed with closed eyes on lactation day 21, due to delayed

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maturation. All F1 pups at the 100 mg/kg/day dose level were euthanized on LD 21 due to

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concerns about their ability to survive without the dam. There were no differences in pup

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viability, survival, clinical observations, or body weight at any other dose level. No differences

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in live born or viability indices, litter size, or sex ratio were observed at any dose level.

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Among F1 males and females selected for further evaluation after weaning, there were no effects or statistically significant differences in the age at attainment of preputial separation or Page 11 of 39

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vaginal patency, body weight parameters, and nutritional parameters; and no clinical

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abnormalities or mortality, at dose levels ≤25 mg/kg/day, which was the highest level evaluated

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due to the early termination of the 100 mg/kg/day group (Table 3).

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3.5 Hematology in P1 mice

6:2 FTOH exposure at 100 mg/kg/day resulted in hematology findings for both red and

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white cell parameters in males and females (Figure 3). Group mean red cell mass parameters

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(RBC, HGB, and HCT) were minimally to mildly decreased by up to 11 and 17%, in males and

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females, respectively (statistically significant for most parameters). In females, but not males,

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red cell mass changes were associated with an increase in ARET (132% above controls).

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Additional parameters reflecting these changes in red blood cell mass included minimal (less

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than 10% change compared to controls) but statistically significant decreased MCV in males (4%

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below control), increased RDW in males (8% above control), and decreased MCH in females

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(6% below control). Total white blood cells were increased by 41% (males) and 110%

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(females), and were associated with statistically significant increases in females (but not males)

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in absolute neutrophil, lymphocyte, and monocyte counts (data not shown).

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No effects or statistically significant differences in hematology parameters were observed in males or females at dose levels ≤25 mg/kg/day.

3.6 Clinical chemistry in P1 mice

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Effects on liver-associated clinical chemistry parameters were observed in males and

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females administered 100 mg/kg/day (Figure 4). Changes for AST, ALT, ALKP, SDH, and

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TBA were generally mild to moderate in males and moderate to severe in females, as group

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mean liver parameters were increased approximately 2.5- to 5-fold compared to controls in Page 12 of 39

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males, whereas in females, most parameters were increased by greater than 5-fold and by as

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much as 19-fold (TBA in females) compared to controls. Although not statistically significant,

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minimal increases in total BILI in these groups may also have been 6:2 FTOH-related.

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Elevations in some liver parameters (AST, ALT, SDH, and TBA, but not ALKP or BILI) in one

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25 mg/kg/day female were associated with microscopic lesions in the liver and were attributed to

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6:2 FTOH exposure.

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Effects on other clinical pathology parameters in males and females at 100 mg/kg/day

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included decreases in the following (data not shown): BUN, decreased by 28% and 18% in males

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and females, respectively; CREA, decreased by 37 and 20%, respectively; and CHOL (males

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only; decreased by 35%). In addition, potassium was minimally higher in males and females at

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100 mg/kg/day (23 and 15% above the controls, respectively; statistically significant in males

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only)

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There were no effects on clinical chemistry parameters in males at dose levels ≤25 mg/kg/day, or in females at dose levels ≤5 mg/kg/day.

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3.7 Organ weights in P1 mice

Primary organ weight effects were observed in the liver of males and females at 100

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mg/kg/day, and in the kidneys of males at 100 mg/kg/day (Tables 4 and 5). In addition,

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reproductive organ weight changes occurring secondary to body weight decrements were

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observed in male and female reproductive organs (Tables 4 and 5).

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Group mean liver weight parameters were increased in both sexes (only liver weight

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relative to body weight was statistically significant), although these increases were greater in

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females compared to males (liver weight relative to body weight was increased 13% and 24%

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above control, in males and females, respectively). Page 13 of 39

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Group mean kidney weight parameters were increased in males at 100 mg/kg/day (up to

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25% above controls; statistically significant). These increases were not associated with relevant

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changes in kidney-related clinical pathology parameters or with microscopic changes in the

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kidney. Kidney weight relative to body weight was minimally higher in females at 100

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mg/kg/day (11% above control; statistically significant). However, there were no statistically

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significant changes in other kidney weight parameters, and no treatment-related changes in

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kidney-related clinical pathology parameters or kidney histopathology. Therefore, this change in

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female mice was considered to be unrelated to 6:2 FTOH administration.

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Organ weight changes occurring secondary to body weight decrements were observed in

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male and female reproductive organs. Absolute and relative ovarian and uterine weight

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parameters were moderately to markedly decreased in females at 100 mg/kg/day. Ovarian

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weights were decreased by up to 24% compared to controls, and uterine weights were decreased

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by up to 56% compared to controls (statistically significant for most weight parameters). Testes

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weight relative to body weight was statistically increased (15% above control) in males at 100

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mg/kg/day.

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males or females at dose levels ≤25 mg/kg/day.

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3.8 Histopathology in P1 mice

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Liver

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Microscopic findings indicative of trophic and toxic effects on the liver were present in

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males and females at 100 mg/kg/day and in low incidences in females at 25 mg/kg/day (Table 6).

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These changes were generally more severe in females and included hepatocellular hypertrophy,

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oval cell hyperplasia, single cell necrosis of hepatocytes, and cystic degeneration (females only). Page 14 of 39

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Minimal microscopic hepatocellular hypertrophy was also present in males at 5 and 25

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mg/kg/day and in females at 5 mg/kg/day. Hepatocellular hypertrophy was most prominent in centrilobular regions and was

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characterized by enlarged hepatocytes containing increased amounts of pale to brightly

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eosinophilic granular cytoplasm and enlarged nuclei which often had prominent and multiple

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nucleoli. Hyperplastic oval cells, small spindiloid cells containing fusiform to oval nuclei and

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scant basophilic cytoplasm, radiated from periportal regions into the surrounding parenchyma

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and were often admixed with inflammatory cell infiltrates. Single cell necrosis (or apoptosis) of

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hepatocytes was most conspicuous in periportal regions and was characterized by individual

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shrunken, hypereosinophilic cells containing pyknotic nuclei. Focal to multi-focal areas of cystic

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degeneration, likely resulting from dropout of degenerate hepatocytes, were characterized by

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variably-sized cystic areas containing flocculent to homogenous eosinophilic material, often

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admixed with inflammatory cell infiltrates.

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336 337 338

Teeth

Ac ce p

No microscopic effects were observed in the liver of males or females at 1 mg/kg/day.

335

6:2 FTOH-related changes in the incisor teeth, consistent with fluoride exposure, were

339

present in males and females at 100 mg/kg/day. These changes included degeneration and

340

atrophy of ameloblastic epithelium, accentuation of the normal laminar pattern of dentin and an

341

increase in observed incomplete decalcification of enamel and/or dentin. Degeneration and

342

atrophy of ameloblasts was characterized by segmental disorganization and attenuation of

343

ameloblastic epithelium of the incisor teeth. Lamination of dentin was characterized by the

344

presence of concentric basophilic rings within the dentin of these teeth. Incomplete

345

decalcification of enamel and dentin was characterized by an increase in the observed presence Page 15 of 39

Page 15 of 50

of basophilic, mineralized debris in the enamel space of the incisor between the dentin and the

347

gingiva. Normally, this material is lost during processing of slides and is not visible, but it is

348

observed sporadically in control mice due to vagaries in processing. However, this finding was

349

observed at a higher incidence and extent in males and females administered 100 mg/kg/day.

350

Incomplete decalcification of nasal bones, also consistent with fluoride exposure, was observed

351

in some animals at the 100 mg/kg/day dose level. There were no adverse changes in the teeth of

352

males or females at dose levels ≤25 mg/kg/day.

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346

354 355

Female Reproductive Tract and Mammary Gland

an

353

Eight of 11 nursing dams in the 100 mg/kg/day group that survived to the scheduled euthanasia at weaning were in anestrus. No dams in the control or other treated groups were

357

anestral. Anestrus was characterized by marked thinning of the vaginal mucosal epithelium

358

(which was often only a single cell layer thick), uterine involution and atrophy of ovarian

359

interstitial cells. In the mammary glands of nursing dams in the 100 mg/kg/day group that

360

survived to weaning, microscopic changes included decreased milk secretion (in 8/11 dams),

361

characterized by lactational hypertrophy and hyperplasia of glandular epithelium consistent with

362

an actively nursing dam, but with few or no alveoli containing proteinaceous secretion, and

363

apparent mineralization (deep basophilic staining) of glandular secretion (in 11/11 dams).

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364

No treatment-related changes in reproductive tract histology, estrus cyclicity or

365

mammary glands were observed at dose levels ≤25 mg/kg/day, where body weight and

366

nutritional parameters in both dams and offspring were similar to controls.

367 368

3.9 Gross pathology in F1 mice

Page 16 of 39

Page 16 of 50

There were no 6:2 FTOH-related gross findings in F1 pups or weanlings at dose levels

369 370

≤100 mg/kg/day, nor in F1 adults at dose levels ≤25 mg/kg/day (the highest level evaluated due

371

to the early termination of the 100 mg/kg/day group).

374

4.0

DISCUSSION

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372

The primary objective of this study was to evaluate the oral systemic repeated-dose and reproductive toxicity of 6:2 FTOH in CD-1 mice. Results from the present evaluation in mice, in

376

conjuction with previously published results using CD rats (O’Connor et al., 2014; Serex et al.,

377

2014), provide a basis for comparison between commonly used outbred strains of two rodent

378

species.

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379

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4.1 Repeated-dose systemic and reproductive toxicity of 6:2 FTOH in mice At 100 mg/kg/day, mortality was observed in 1/15 male and 2/15 female mice, as well as

te

380

clinical signs of behavioral and physiological dysfunction, and reductions in body weight and

383

nutritional parameters. Reductions in body weight were 5% in P1 males and 20% in P1 females.

384

Alterations in red blood cell parameters at this dose level may be secondary to the body weight

385

reductions (Everds et al. 2013). In general, the alterations in red blood cell parameters were

386

more severe in females than in males, and were associated with a regenerative response only in

387

females, which corresponded to the sex difference in body weight reductions. Increased white

388

blood cell counts at 100 mg/kg/day in males and females likely represent an inflammatory

389

response to liver toxicity. Elevated clinical chemistry parameters indicative of hepatocellular

390

injury (AST, ALT, ALP, SDH, and TBA) were greater in females than males at 100 mg/kg/day,

391

and were also observed in one female (and no males) at 25 mg/kg/day.

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Page 17 of 39

Page 17 of 50

Correspondingly, histopathologic alterations in the liver were more severe in females

392

than males. While liver weights were increased in both sexes at 100 mg/kg/day, adverse

394

microscopic liver effects (including singe cell necrosis, cystic degeneration, oval cell hyperplasia

395

and increased pigment) were observed in females at dose levels ≥25 mg/kg/day and males at 100

396

mg/kg/day. In addition, decreases in BUN, CREA, and/or CHOL were observed in males and

397

females at 100 mg/kg/day; the decreases may be secondary to the liver toxicity observed at this

398

dose level (Hall and Everds, 2008) but may also be in response to food consumption reduction

399

and/or to hepatic enzyme induction (Amacher et al., 1998). Hepatocellular hypertrophy was

400

observed at dose levels ≥5 mg/kg/day in both sexes, but was considered non-adverse when it

401

occurred in the absence of correlative liver weight changes or histologic evidence of

402

hepatotoxicity, based on industry standards and regulatory guidance (ECETOC, 2002; U.S. EPA,

403

2002).

cr

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The findings of hepatocellular hypertrophy were morphologically consistent with

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404

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393

peroxisome proliferation and, along with increased mitotic figures, were consistent with the

406

known trophic effects produced in the liver of rodents by peroxisome proliferator-activated

407

receptor (PPAR) alpha agonists (Peters et al., 2005).

408

included single cell necrosis and cystic degeneration. Hyperplastic oval cells likely represent a

409

regenerative response to liver cell injury and are considered to arise from terminal ductule

410

epithelial cells (canal of Hering cells) within periportal regions of the liver (Thoolen et al., 2010).

411

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405

Microscopic evidence of hepatotoxicity

6:2 FTOH has been shown to rapidly metabolize with formation of fluoride (Gannon et

412

al., 2010; Gannon et al., 2012). Microscopic alterations of incisor teeth in both male and female

413

mice at 100 mg/kg/day included degeneration and atrophy of ameloblastic epithelium (an

414

adverse change), lamination of dentin, and incomplete decalcification of enamel and/or dentin.

415

Changes in the ameloblastic epithelium and lamination of dentin have been previously described Page 18 of 39

Page 18 of 50

in the rat incisor following fluoride exposure (Yeager, 1966; Walton and Eisenmann, 1975;

417

Fejerskov et al., 1979). Incomplete decalcification of teeth and bone are likely the result of

418

fluoride-associated resistance to the acid decalcification procedure used in this study, as fluoride

419

can reduce the acid solubility of mineral in teeth and bones (Jenkins, 1963; Aoba, 1997) and as

420

such, this finding was considered to be indicative of exposure to a metabolized fluoride, but non-

421

adverse.

cr

Reproductive organ weight parameters and/or histology were altered in males and

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422

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416

females as secondary effects of decrements in body weight and nutritional parameters at 100

424

mg/kg/day. In males, an increased ratio of testes weight to body weight was attributed to body

425

weight loss, as testes weights are known to be stable with moderate levels of body weight loss

426

(Chapin et al., 1993) and there were no correlative histologic findings in the testes. In females at

427

this dose level, ovarian and uterine weight parameters were decreased, which corresponded to

428

microscopic findings including anestrus with associated atrophic changes in the reproductive

429

tract, and secretory depletion in the mammary gland. Anestrus and the correlative microscopic

430

changes in the female reproductive tract are common non-specific findings associated with

431

reduced food intake and stress (Yuan and Foley, 2002). Secretory depletion in the mammary

432

gland is likely the result of increased duration of nursing in the undernourished pups at 100

433

mg/kg/day, as undernourished rat pups from diet-restricted dams have increased duration of

434

suckling compared to controls, especially toward the end of lactation (Riul et al., 1999).

435

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423

The following changes were attributed to 6:2 FTOH exposure but non-adverse based on

436

lack of association with organ injury or evidence of decreased function, as described above:

437

increased kidney weights (100 mg/kg/day males), incomplete decalcification of enamel and

438

dentin (25 mg/kg/day females), and minimal hepatocellular hypertrophy (5 mg/kg/day males and

439

females; 25 mg/kg/day males). Page 19 of 39

Page 19 of 50

440

Among F1 pups, adverse effects at 100 mg/kg/day included mortality (45% reduction in lactation index), reduced body weights (65% reduction), and clinical signs of delayed

442

maturation. The failure to thrive and delayed maturation of the pups were likely a result of the

443

overt systemic toxicity observed in the dams.

444

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441

For mice, the no-observed-adverse-effect level (NOAEL) for systemic repeated-dose toxicity was 25 mg/kg/day (males) and 5 mg/kg/day (females), based on effects at higher doses

446

on mortality, clinical observations, body weight, nutritional parameters, hematology (red and

447

white blood cell), clinical chemistry (liver-related), liver weights, and histopathology (liver,

448

teeth, reproductive tract, and mammary gland). However, 6:2 FTOH was not a selective

449

reproductive toxicant; any effects observed in offspring occurred at dose levels that induced

450

mortality and severe toxicity in maternal animals. The NOAEL for reproductive toxicity was

451

100 mg/kg/day; no effects on reproductive outcome were observed at any dosage. The NOAEL

452

for viability and growth of the offspring was 25 mg/kg/day, based on clinical signs of delayed

453

maturation in pups, and reductions in pup survival and pup body weight during lactation at 100

454

mg/kg/day.

456

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Ac ce p

455

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445

4.2 Comparison of 6:2 FTOH toxicity in rats and mice

457

The results of the present study can be compared with previous oral repeated-dose and

458

reproduction studies in CD rats with 6:2 FTOH (Serex et al. 2014, O’Connor et al., 2014) and

459

reflects the toxicological action and effects of 6:2 FTOH and its metabolites. The severity and

460

range of effects were generally greater in mice, especially with regard to liver

461

histopathology/function and pup growth/viability. However, the overall NOAELs for systemic

462

toxicity, reproductive toxicity, and offspring viability/growth were consistent between species

463

(Table 7), and 6:2 FTOH was not a selective reproductive toxicant in either species. Page 20 of 39

Page 20 of 50

464

Among P1 adults, the NOAEL for mortality and clinical observations was 25 mg/kg/day in both species (Table 7). However, the NOAEL for body weight loss during lactation was

466

higher in rats than mice, and the magnitude of the reduction at 100 mg/kg/day in mice exceeded

467

that at 250 mg/kg/day in rats. The NOAEL for liver-related clinical chemistry changes and

468

associated microscopic liver findings was 5 mg/kg/day in both species, although the extent of

469

hepatocellular injury was more pronounced in mice. The magnitude of differences in clinical

470

chemistry parameters and the incidences of histopathologic liver findings at 100 mg/kg/day in

471

mice exceeded those at 250 mg/kg/day in rats. Correspondingly, white blood cell alterations,

472

which may be associated with liver inflammation due to hepatotoxicity, were observed at 100

473

mg/kg/day in mice, but were not reported in rats at dose levels ≤250 mg/kg/day. Histopathologic

474

evidence of liver toxicity in the rat (single cell necrosis, biliary and oval cell hyperplasia;

475

hepatocyte vacuolation and peri-portal-associated inflammation) was reported for only a few

476

animals (at ≥125 mg/kg/day in males and at ≥25 mg/kg/day in females), and generally with

477

minimal severity (Serex et al., 2014; DuPont, unpublished data). In contrast, liver toxicity in the

478

mouse (singe cell necrosis, cystic degeneration, oval cell hyperplasia, oval cell-associated

479

mononuclear infiltrate and increased pigment) generally affected most animals of both sexes at

480

100 mg/kg/day with minimal to mild severity. Microscopic alterations in the incisor teeth were

481

observed in mice at 100 mg/kg/day whereas similar effects were only reported at 250 mg/kg/day

482

in rats. Microscopic findings in reproductive organs (anestrus with associated atrophic changes

483

in the reproductive tract, and secretory depletion in the mammary gland) occurring secondary to

484

decrements in body weight and nutritional parameters were observed at 100 mg/kg/day in mice,

485

but were not reported in rats at dose levels ≤250 mg/kg/day. This difference between the two

486

species was consistent with the greater difference in maternal body weight during lactation, in

487

mice compared to rats.

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Page 21 of 39

Page 21 of 50

488

Among F1 pups, the NOAEL for mortality, clinical observations, and reduced body weights was 25 mg/kg/day in both species. The magnitude of the reductions in lactation index

490

and pup body weight at 100 mg/kg/day in mice exceeded those observed in rats at 250

491

mg/kg/day, and is likely a result of the species difference in maternal body weight during

492

lactation.

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489

493

495

4.3 Comparison of 6:2 FTOH toxicity with other fluorotelomer alcohols and metabolites

us

494

Reproductive or systemic toxicity data are available for 8:2 FTOH; a fluoroalkylethanol mixture, F(CF2CF2)nCH2CH2OH (n = 3 to 6), including 30%(w/w) 6:2 FTOH; and PFHxA, a

497

principal metabolite of 6:2 FTOH. Toxicity data for these related chemicals are consistent with

498

data reported in both rats and mice for 6:2 FTOH with regard to NOAELs, the types of effects,

499

and the lack of selective reproductive toxicity where applicable, i.e. any effects on offspring

500

viability and growth were only observed at concentrations that were also associated with

501

systemic toxicity. Reproductive evaluations of the fluoroalkylethanol mixture and PFHxA in CD

502

rats yielded NOAELs of 20 to 50 mg/kg/day for systemic toxicity, greater than 250 mg/kg/day

503

(the highest dose evaluated) for reproductive toxicity, and 25 to 100 mg/kg/day for offspring

504

viability and growth (Mylchreest et al., 2005b; Loveless et al., 2009; O’Connor et al., 2014).

505

Subchronic evaluations of 8:2 FTOH, the fluoroalkylethanol mixture, and PFHxA in CD rats

506

yielded NOAELs ranging from 5 to 25 mg/kg/day, based on effects at higher doses on body

507

weight, clinical observations, liver and/or kidney weights, histopathology (liver, kidney, thyroid,

508

or teeth), red blood cell and liver-related clinical chemistry parameters, and urinary or plasma

509

fluoride (Ladics et al., 2005; Ladics et al., 2008; Loveless et al., 2009).

510 511

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496

8:2 FTOH has been evaluated for developmental and hepatotoxicity in mice (CD-1 or ddY strains) in addition to CD rats (Kudo et al., 2005; Mylchreest et al., 2005a; Henderson and Page 22 of 39

Page 22 of 50

Smith, 2007; Ladics et al., 2008). The studies in mice differed from those in rats with respect to

513

basic study design elements (e.g., number of exposures, diet vs. gavage), and they did not

514

include a full evaluation of reproductive toxicity. Nonetheless, the differences between species

515

for 8:2 FTOH toxicity were similar to those presently reported for 6:2 FTOH, i.e., mice were

516

generally more sensitive. Compared with rats, 8:2 FTOH exposure in mice resulted in

517

hepatomegaly at a lower dose level and developmental toxicity (neonatal mortality and

518

malformations in mice) with greater severity at a lower dose, although the effects on the fetus in

519

both species were only observed at levels that induced maternal toxicity. The greater sensitivity

520

of mice to hepatotoxic effects has also been demonstrated in evaluations of perfluorobutyrate

521

(PFBA) and perfluorooctanoate (PFOA), which are metabolites of 6:2 FTOH and 8:2 FTOH,

522

respectively. Exposure to PFBA and PFOA resulted in the same or lower NOAELs for

523

hepatotoxicity in CD-1 mice compared with CD rats, while the severity of

524

hepatomegaly/hypertrophy and the range of histopathologic effects (e.g. necrosis, increased

525

mitotic figures) was generally greater in mice (Loveless et al., 2006; Loveless et al., 2008; Das et

526

al., 2008; Butenhoff et al., 2012). These comparisons are generally limited to one strain of each

527

species (CD-1 mice and CD rats). The only other strain included in the discussion above was the

528

ddY strain of mouse (Japan), which yielded results that were consistent with those from the CD-

529

1 strain (Kudo et al., 2005).

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530

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512

Mechanisms underlying the greater sensitivity of mice to hepatotoxic effects are not

531

certain, but several proposed explanations include possible species differences in peroxisome

532

proliferation or in kinetic properties of metabolites. Induction of peroxisome proliferation in

533

rodents has been confirmed for 8:2 FTOH, PFBA, and PFOA (Kudo et al., 2005; Loveless et al.,

534

2006; Das et al., 2008), and may be presumed for 6:2 FTOH on the basis of histologic effects

535

described in section 4.1 and beta oxidation measurements in one of its principal metabolites, 5:3 Page 23 of 39

Page 23 of 50

acid (MacKenzie et al., 2013). To the extent that peroxisome proliferation may be responsible

537

for any species differences in 6:2 FTOH toxicity, the relevance for humans may be limited,

538

because humans tend to be resistant to such effects, in contrast to rats and mice (Bentley et al.,

539

1993). Loveless et al. (2006) noted that species differences in PFOA toxicity may be a result of

540

additional mechanisms as well, due to the inconsistent correlation between liver weight increases

541

and peroxisomal beta oxidation.

cr

Henderson and Smith (2007) suggested that the sensitivity of mice to 8:2 FTOH may be

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542

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536

attributed to the role of terminal metabolites such as perfluorooctanoic acid (PFOA). This

544

assertion formed a basis for proposed hazard classifications for 8:2 FTOH which were not

545

adopted by the ECHA Committee for Risk Assessment (Climate & Pollution Agency, Norway,

546

2012; ECHA, 2013), due to a wider body of kinetic, molecular, and epidemiologic data that did

547

not support this claim (Lau et al., 2006; Nabb et al., 2007; Calafat et al., 2007; Haug et al., 2009;

548

Haug et al., 2010; Haug et al., 2011; US CDC, 2012; Himmelstein et al., 2012b). In addition,

549

these classification arguments pertaining to longer chain fluorochemicals are not directly

550

applicable to 6:2 FTOH, because of differences between short chain and longer chain

551

metabolites. PFHxA (the analogous 6-carbon metabolite from 6:2 FTOH) is eliminated from

552

plasma 40-80 times faster than PFOA in humans, rats, and mice (Olsen et al., 2007; Gannon et

553

al., 2011; Russell et al., 2013), and the hepatotoxicity is correspondingly lower in rats and mice

554

as well (Perkins et al., 2004; Loveless et al., 2009).

555

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543

Species differences in FTOH toxicity may also be due to possible kinetic differences in

556

other metabolites such as fluorotelomer unsaturated aldehydes (FTUALs), which are potential

557

precursors to perfluorocarboxylic acids (PFCAs) such as PFOA and PFHxA. Evaluations of

558

toxicokinetics, protein reactivity, cytotoxicity, and acute aquatic toxicity have indicated that

559

FTUALs are generally more toxic than the PFCAs, and have been suggested to be responsible Page 24 of 39

Page 24 of 50

for rat liver lesions following FTOH exposure (Fasano et al., 2006; Martin et al., 2006;

561

MacDonald Phillips et al., 2007; Rand et al., 2012a, 2012b, and 2013). Further evaluation would

562

be required to determine species differences in kinetics of FTUALs and other FTOH metabolites.

563

Repeated-dose evaluations of intermediate metabolites may not be possible because of their

564

relative instability.

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560

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5:3 acid is another 6:2 FTOH metabolite that has been evaluated in mammalian and

565

aquatic species for biopersistence, acute oral toxicity, and two week repeated-dose toxicity. The

567

extent of acute and systemic toxicity was considered consistent with previously reported data for

568

6:2 FTOH and indicated low potential for bioaccumulation in mammals (MacKenzie et al.,

569

2013). Although 5:3 acid has not been specifically evaluated for reproductive or subchronic

570

toxicity, the results of the present evaluation of 6:2 FTOH represents the combined effects of the

571

parent chemical and all metabolites, including the principal metabolites PFHxA and 5:3 acid, and

572

intermediates such as FTUALs.

575

5.0

CONCLUSIONS

Ac ce p

574

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573

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566

CD-1 mice were generally more sensitive than CD rats with respect to systemic toxicity

576

from oral administration of 6:2 FTOH, and this was likely responsible for the more severe effects

577

observed in the offspring (i.e., pup growth and viability) at doses where systemic effects were

578

observed. The liver was the primary target organ in both species, evidenced by increased liver

579

size, trophic and toxic histopathologic effects, and elevations in clinical chemistry parameters

580

that were consistent with hepatotoxicity. Mortality, clinical abnormalities, red blood cell

581

changes, and reductions in male body weights were consistent across species, but the reduction

582

in P1 female body weight during lactation was markedly greater in mice than in rats.

583

Correspondingly, changes in the female reproductive tract and mammary gland secondary to Page 25 of 39

Page 25 of 50

body weight decrements were observed only in mice, and the magnitude of the reductions in

585

lactation index and pup body weight were greater in mice than in rats. While the severity of the

586

effects was generally greater in mice than rats, the overall NOAELs were identical in both

587

species, 5 mg/kg/day for systemic toxicity and 25 mg/kg/day for offspring viability/growth. 6:2

588

FTOH was not a selective reproductive toxicant in either species; no effects on reproductive

589

outcome occurred at any dose level, and any effects observed in offspring occurred at dose levels

590

that induced mortality and severe toxicity in maternal animals. The present evaluation of 6:2

591

FTOH in CD-1 mice corroborates the benchmark dose of 5 mg/kg/day previously reported for

592

6:2 FTOH in CD rats. Together, these studies reflect the toxicological action and effects of 6:2

593

FTOH and its metabolites in rats and mice.

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584

594

CONFLICT OF INTEREST

596

The authors are employed by DuPont, which is a manufacturer of 6:2 FTOH.

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597 598

ACKNOWLEDGEMENTS

599

The authors would like to thank the technicians who conducted the study and our colleagues who

600

reviewed the manuscript.

601 602

Page 26 of 39

Page 26 of 50

603

FIGURE LEGENDS

604

Figure 1. P1 In-Life Parameters During Lactation

606

Mean values are displayed. # Significantly different (p < 0.05) from control by Dunnett’s Test.

607

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605

Figure 2. F1 Mortality and Pup Weights During Lactation

609

Mean values are displayed. # Significantly different (p < 0.05) from control by Dunnett’s Test.

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608

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610

Figure 3. P1 Hematology Parameters

612

Groups 1, 2, 3, 4, and 5 correspond to doses of 0, 1, 5, 25, and 100 mg/kg/day, respectively.

613

Mean values and standard error bars are displayed. Units: (a) x106/µL; (b) g/dL; (c) %; (d)

614

x103/µL. # Significantly different (p < 0.05) from control by Dunnett’s Test.

d te

615

M

611

Figure 4. P1 Clinical Chemistry Parameters

617

Groups 1, 2, 3, 4, and 5 correspond to doses of 0, 1, 5, 25, and 100 mg/kg/day, respectively.

618

Mean values and standard error bars are displayed. Units: (a) U/L; (b) mol/L; (c) mg/dL. #

619

Significantly different (p < 0.05) from control by Dunnett’s Test.

620 621

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622

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623

Amacher, D.E., Schomaker, S.J., and Burkhardt, J.E., 1998. The relationship among microsomal

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Aoba, T., 1997. The effect of fluoride on apatite structure and growth. Crit Rev Oral Biol Med,

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Bentley, P., Calder, I., Elcombe, C., Grasso, P., Stringer, D., Weigand, H.J., 1993. Hepatic

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Liedera, P.H., van Otterdijke, F.M., Wallace, K.B., 2012. Toxicological evaluation of

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Calafat, A.M., Wong, L-Y, Kuklenyik, Z., Reidy, J.A., Needham, L.L., 2007. Polyfluoroalkyl

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Chapin, R., Gulati, D., Barnes, L., Teague, J., 1993. The effects of feed restriction on

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Chengelis, C.P., Kirkpatrick, J.B., Radovsky, A., Shinohara, M., 2009. A 90-day repeated dose

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observational battery and motor activity determinations). Reprod Toxicol, 27 (3-4), pp. 342-351.

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Das, K.P., Grey, B.E., Zehr, R.D., Wood, C.R., Butenhoff, J.I., Chang, S-C., Ehresman, D.J.,

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Tan, Y-M., Lau, C., 2008. Effects of perfluorobutyrate exposure during pregnancy in the mouse.

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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ip t

Tables 1-7

0

1

5

us

Dose (mg/kg/day)

cr

Table 1. P1 In-Life Parameters

25

100

14 42.4 (2.9) 11.6 (2.3) 5.8 (0.6)

15 42.9 (3.2) 12.1 (3.7) 5.9 (0.5)

15 42.2 (2.9) 11.6 (2.2) 5.8 (0.5)

15 40.4 (2.2) 11.0 (1.7) 5.6 (0.5)

14 40.4 (3.2) 10.2 (2.9) 5.4 (0.5)

Females - premating Body weight (Day 14) Body weight gain (Day 0-14) Daily food consumption (Day 0-14)

15 27.3 (1.3) -0.1 (0.9) 4.6 (0.5)

15 27.7 (1.4) 0.4 (0.9) 4.6 (0.5)

15 27.7 (1.5) 0.3 (1.7) 4.9 (0.5)

15 27.9 (1.8) 0.3 (1.2) 4.6 (0.5)

14 27.4 (1.1) -0.1 (1.1) 5.0 (0.5)

Females - gestation Body weight (GD 17) Body weight gain (GD 0-17) Daily food consumption (GD 0-17)

14 52.1 (4.0) 25.2 (3.1) 6.4 (0.7)

14 54.7 (5.5) 27.6 (4.4) 6.3 (0.6)

14 54.0 (4.1) 26.9 (3.6) 6.5 (0.5)

14 54.5 (4.6) 26.7 (3.5) 6.3 (0.7)

10 53.4 (3.9) 24.4 (4.9) 6.3 (0.4)

14 41.5 (4.4) 6.0 (3.1) 17.7 (1.5)

15 39.9 (3.7) 4.9 (2.8) 17.6 (1.6)

14 40.4 (3.4) 5.1 (3.3) 17.3 (1.4)

9 35.7 (2.9) 1.7 (2.9) 10.5 (1.7)#

ce pt

ed

M an

Males Final body weight (Day 105) Body weight gain (Day 0-105) Daily food consumption (Day 0-70)

Females - lactation Body weight (LD 21) Body weight gain (LD 0-21) Daily food consumption (LD 0-14)

13 39.2 (2.8) 3.8 (3.2) 17.6 (2.3)

Ac

Numbers of animals completing each phase are listed. Data are presented as mean (standard deviation). Units are grams (body weight parameters) and grams/day (food consumption). # Significantly different (p < 0.05) from control by Dunnett’s Test.

Page 1 of 7

Page 44 of 50

ip t 1

15 14 14 15 100 100 100 93.7 99.0

15 15 14 14 100 93.3 100 100 100

5

us

Number of females paired Number of females mated Number of pregnant females Number of females that littered Mating index (%)a Fertility index (%)b Gestation index (%)c Viability index (%)d Lactation index (%)e

0

M an

Dose (mg/kg/day)

cr

Table 2. Reproductive Parameters and F1 Litter Data

15 14 14 15 100 100 100 99.5 100

(1.3) (0.1) (2.5) (2.7) (2.7) (15.9)

100

15 15 14 14 100 93.3 100 100 99.1 2.6 19.0 12.1 11.1 11.0 40.7

14 11 11 12 100 100 100 90.3 54.5 # (1.8) (0.3) (1.9) (1.7) (1.8) (14.6)

2.5 18.9 11.2 11.6 11.4 48.8

(1.6) (0.5) (3.3) (1.6) (1.3) (19.9)

Ac

ce pt

ed

Precoital interval (days) 2.0 (1.2)f 2.0 (1.1) 2.2 Mean gestation length (days) 18.9 (0.3) 18.8 (0.4) 19.0 Implantations 11.7 (1.5) 12.1 (1.6) 11.5 Number of pups born 11.0 (2.0) 11.5 (1.5) 11.3 Number of pups born alive 11.0 (2.0) 11.5 (1.5) 11.3 Sex ratio (% males) 52.0 (20.6) 51.6 (8.5) 47.6 a Number copulated/number paired b Number pregnant/number copulated c Number of litters with at least 1 live pup/number of litters d Number of pups alive on day 4/number of pups born alive e Number of live pups on lactation day 21/number of live pups on day 4 post-culling f Data are presented as mean (standard deviation) # Significantly different (p < 0.05) from control by Dunnett’s Test

25

Page 2 of 7

Page 45 of 50

ip t 0

1

5

us

Dose (mg/kg/day)

cr

Table 3. F1 In-Life Parameters

25

13 18.0 (2.1) 5.4 (0.6) 28.3 (1.5)

14 18.0 (2.3) 5.7 (0.5) 27.5 (1.2)

15 17.5 (1.5) 5.4 (0.4) 27.3 (1.7)

14 18.2 (1.7) 5.4 (0.5) 27.9 (1.5)

F1 females Body weight gain (PND 21-40) Daily food consumption (PND 21-40) Age at vaginal patency achievement

13 11.2 (1.2) 4.8 (0.4) 29.8 (2.2)

14 11.0 (1.4) 4.9 (0.4) 29.4 (2.6)

14 11.8 (1.2) 5.1 (0.4) 29.2 (2.8)

14 11.2 (1.9) 4.9 (0.5) 30.2 (2.2)

M an

F1 males Body weight gain (PND 21-40) Daily food consumption (PND 21-40) Age at preputial separation achievement

Ac

ce pt

ed

Numbers of animals completing each phase are listed. Data are presented as mean (standard deviation). Units are grams (body weight gain), grams/day (food consumption), and post-natal days (preputial separation or vaginal patency achievement). There were no statistically significant differences (p < 0.05).

Page 3 of 7

Page 46 of 50

ip t 1 (15)

41.9 2.162 0.659 0.123 0.235

(3.1) (0.235) (0.117) (0.014) (0.027)

Relative Liver Kidneys Epididymides Testes

5.159 4.564 0.296 0.565

(0.402) (0.207) (0.046) (0.076)

54.8 2.140 0.692 0.125 0.253

(2.9) (0.219) (0.101) (0.014) (0.031)

5.000 1.613 0.294 0.593

(0.417) (0.175) (0.038) (0.085)

ed

Absolute Terminal body weight Liver Kidneys Epididymides Testes

5 (15)

us

0 (14)

25 (15)

100 (14)

42.0 2.054 0.6613 0.125 0.258

(2.6) (0.202) (0.054) (0.001) (0.040)

40.6 2.093 0.710 0.121 0.230

(2.2) (0.162) (0.089) (0.011) (0.021)

39.4 2.296 0.773 0.123 0.254

(2.9) (0.264) (0.123)# (0.016) (0.028)

4.903 1.576 0.299 0.616

(0.483) (0.191) (0.031) (0.094)

5.163 1.748 0.298 0.568

(0.353) (0.190) (0.033) (0.066)

5.817 1.959 0.313 0.647

(0.368)# (0.260)# (0.048) (0.086)#

M an

Dose (mg/kg/day) No. Examined

cr

Table 4. P1 Organ Weights (Males)

Ac

ce pt

Data are presented as mean (standard deviation). Absolute weights are reported in grams; relative weights are reported as percent terminal body weight. # Significantly different (p < 0.05) from control by Dunnett’s Test.

Page 4 of 7

Page 47 of 50

0 (13)

ip t

(2.8) (0.329) (0.027) (0.0045) (0.131)

40.6 3.070 0.525 0.0370 0.202

(5.7) (0.605) (0.090) (0.0088) (0.068)

7.162 1.316 0.081 0.675

(0.604) (0.086) (0.012) (0.334)

7.512 1.293 0.093 0.515

(0.774) (0.113) (0.025) (0.211)

25 (15)

100 (10)

39.9 3.005 0.516 0.0342 0.198

(3.7) (0.418) (0.038) (0.0055) (0.058)

39.8 3.048 0.546 0.0355 0.203

(4.2) (0.526) (0.059) (0.0095) (0.068)

35.4 3.162 0.515 0.0244 0.118

(2.9) (0.640) (0.051) (0.0032)# (0.069)#

7.510 1.296 0.086 0.505

(0.537) (0.081) (0.017) (0.178)

7.624 1.376 0.089 0.518

(0.911) (0.114) (0.023) (0.198)

8.873 1.454 0.069 0.332

(1.341)# (0.080)# (0.010) (0.176)#

M an

Relative Liver Kidneys Ovaries Uterus

5 (15)

39.2 2.809 0.514 0.0316 0.264

ed

Absolute Terminal body weight Liver Kidneys Ovaries Uterus

1 (15)

us

Dose (mg/kg/day) No. Examined

cr

Table 5. P1 Organ Weights (Females)

Ac

ce pt

Data are presented as mean (standard deviation). Absolute weights are reported in grams; relative weights are reported as percent terminal body weight. # Significantly different (p < 0.05) from control by Dunnett’s Test.

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ip t Male

Female

5

25

100

0

1

(15)

(15)

(15)

(15)

(15)

(15)

Hypertrophy, hepatocellular

0

0

9

10

15

Mitotic figures, increased

0

0

0

0

Oval cell hyperplasia

0

0

0

Cystic degeneration

0

0

Single cell necrosis

0

Infiltrate, mononuclear (oval cell associated) Pigment, increased

25

100

(15)

(15)

(15)

(15)

0

0

13

12

13

5

0

1

0

1

10

0

15

0

0

0

2

12

0

0

0

0

0

1

1

7

0

0

0

12

2

1

1

1

12

0

0

0

0

15

0

0

0

0

10

0

0

15

0

0

0

0

0

0

0

Ac

ce pt

No. examined

5

us

1

M an

0

ed

Dose (mg/kg/day):

cr

Table 6. P1 Histopathology of Liver

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25 25 5 25 25 25 5 25 25 25

25 25 5 125 5 >250 b 5 125 25 25

ip t us

5 >250 b 25

M an

5 >100b 25

ce pt

Individual parameters: Mortality Adverse clinical observations Liver histopathology Tooth histopathology Hematology (red blood cell) Hematology (white blood cell) Clinical chemistry Maternal body weight (lactation) Pup survival Pup body weights

Ratsa

ed

Overall: Systemic toxicity Reproductive toxicity Pup viability/growth

Mice

cr

Table 7. NOAELs of 6:2 FTOH in rats and mice

Ac

a O’Connor et al., 2014; Serex et al., 2014. b Highest dose evaluated in the study.

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