[Federal Register Volume 77, Number 87 (Friday, May 4, 2012)]
[Notices]
[Pages 26558-26559]
[FR Doc No: 2012-10828]

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DEPARTMENT OF HEALTH AND HUMAN SERVICES

Food and Drug Administration

[Docket Nos. FDA-2010-E-0661 and FDA-2010-E-0662]

Determination of Regulatory Review Period for Purposes of Patent
Extension; JEVTANA

AGENCY: Food and Drug Administration, HHS.

ACTION: Notice.

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SUMMARY: The Food and Drug Administration (FDA) has determined the
regulatory review period for JEVTANA and is publishing this notice of
that determination as required by law. FDA has made the determination
because of the submission of applications to the Director of Patents
and Trademarks, Department of Commerce, for the extension of a patent
which claims that human drug product.

ADDRESSES: Submit electronic comments to http://www.regulations.gov.
Submit written petitions along with three copies and written comments
to the Division of Dockets Management (HFA-305), Food and Drug
Administration, 5630 Fishers Lane, rm. 1061, Rockville, MD 20852.

FOR FURTHER INFORMATION CONTACT: Beverly Friedman, Office of Regulatory
Policy, Food and Drug Administration, 10903 New Hampshire Ave., Bldg.
51, rm. 6284, Silver Spring, MD 20993-0002,<PHONE_NUMBER>.

SUPPLEMENTARY INFORMATION: The Drug Price Competition and Patent Term
Restoration Act of 1984 (Pub. L. 98-417) and the Generic Animal Drug
and Patent Term Restoration Act (Pub. L. 100-670) generally provide
that a patent may be extended for a period of up to 5 years so long as
the patented item (human drug product, animal drug product, medical
device, food additive, or color additive) was subject to regulatory
review by FDA before the item was marketed. Under these acts, a
product's regulatory review period forms the basis for determining the
amount of extension an applicant may receive.
    A regulatory review period consists of two periods of time: A
testing phase and an approval phase. For human drug products, the
testing phase begins when

the exemption to permit the clinical investigations of the drug becomes
effective and runs until the approval phase begins. The approval phase
starts with the initial submission of an application to market the
human drug product and continues until FDA grants permission to market
the drug product. Although only a portion of a regulatory review period
may count toward the actual amount of extension that the Director of
Patents and Trademarks may award (for example, half the testing phase
must be subtracted as well as any time that may have occurred before
the patent was issued), FDA's determination of the length of a
regulatory review period for a human drug product will include all of
the testing phase and approval phase as specified in 35 U.S.C.
156(g)(1)(B).
    FDA recently approved for marketing the human drug product JEVTANA
(cabazitaxel). JEVTANA, in combination with prednisone, is indicated
for treatment of patients with hormone-refractory metastatic prostate
cancer previously treated with a docetaxel-containing treatment
regimen. Subsequent to this approval, the Patent and Trademark Office
received patent term restoration applications for JEVTANA (U.S. Patent
Nos. 5,847,170 and 6,331,635) from Aventis Pharma S.A., and the Patent
and Trademark Office requested FDA's assistance in determining this
patent's eligibility for patent term restoration. In a letter dated
February 11, 2011, FDA advised the Patent and Trademark Office that
this human drug product had undergone a regulatory review period and
that the approval of JEVTANA represented the first permitted commercial
marketing or use of the product. Thereafter, the Patent and Trademark
Office requested that FDA determine the product's regulatory review
period.
    FDA has determined that the applicable regulatory review period for
JEVTANA is 4,250 days. Of this time, 4,171 days occurred during the
testing phase of the regulatory review period, while 79 days occurred
during the approval phase. These periods of time were derived from the
following dates:
    1. The date an exemption under section 505(i) of the Federal Food,
Drug, and Cosmetic Act (the FD&C Act) (21 U.S.C. 355(i)) became
effective: October 30, 1998. FDA has verified the applicant's claim
that the date the investigational new drug application became effective
was on October 30, 1998.
    2. The date the application was initially submitted with respect to
the human drug product under section 505(b) of the FD&C Act: March 31,
2010. FDA has verified the applicant's claim that the new drug
application (NDA) for JEVTANA (NDA 201023) was submitted on March 31,
2010.
    3. The date the application was approved: June 17, 2010. FDA has
verified the applicant's claim that NDA 201023 was approved on June 17,
2010.
    This determination of the regulatory review period establishes the
maximum potential length of a patent extension. However, the U.S.
Patent and Trademark Office applies several statutory limitations in
its calculations of the actual period for patent extension. In its
applications for patent extension, this applicant seeks 1,591 days and
5 years of patent term extension.
    Anyone with knowledge that any of the dates as published are
incorrect may submit to the Division of Dockets Management (see
ADDRESSES) either electronic or written comments and ask for a
redetermination by July 3, 2012. Furthermore, any interested person may
petition FDA for a determination regarding whether the applicant for
extension acted with due diligence during the regulatory review period
by October 31, 2012. To meet its burden, the petition must contain
sufficient facts to merit an FDA investigation. (See H. Rept. 857, part
1, 98th Cong., 2d sess., pp. 41-42, 1984.) Petitions should be in the
format specified in 21 CFR 10.30.
    Interested persons may submit to the Division of Dockets Management
(see ADDRESSES) electronic or written comments and written petitions.
It is only necessary to send one set of comments. However, if you
submit a written petition, you must submit three copies of the
petition. Identify comments with the docket number found in brackets in
the heading of this document.
    Comments and petitions that have not been made publicly available
on http:[sol][sol]www.regulations.gov may be viewed in the Division of
Dockets Management between 9 a.m. and 4 p.m., Monday through Friday.

    Dated: April 16, 2012.
Jane A. Axelrad,
Associate Director for Policy, Center for Drug Evaluation and Research.
[FR Doc. 2012-10828 Filed 5-3-12; 8:45 am]
BILLING CODE 4160-01-P


BODY WEIGHT       EDSTAC Final Report Chapter Five Appendices August 1998 2 P  VAGINAL CORNIFICATION  +?

3DAYUTEROTROPHICINADULTOVX'D RAT PLUS E2 UTERINE WEIGHT WITH AND WITHOUT FLUID

UTERINE HISTOLOGY

BODY WEIGHT

VAGINAL CORNIFICATION

PUBERTAL/ THYROID ASSAY IN WEANLING FEMALE RAT GROWTH

AGE AT VAGINAL OPENING  ++ ++

WEIGHT AT VAGINAL OPENING  ++ ++

SERUM T4     ++

SERUM TSH     +?

UTERINE WEIGHT WITH AND WITHOUT FLUID   +? ¯

OVARIAN WEIGHT   +? ¯

THYROID HISTOLOGY     +

NONREPRODUCTIVE ORGAN WEIGHTS LIVER     LIVER  LIV  7 10DAYHERSHBERGER TYPEASSAYIN CASTRATED MALE RAT SEMINALVESICLEWEIGHT, WITH AND WITHOUT FLUID

VENTRAL PROSTATE WEIGHT

LEVATOR ANI PLUS BULBOCAVERNOSUS WEIGHT

GROWTH     +?

NONREPRODUCTIVE ORGAN WEIGHTS LIVER     LIVER  LIV  7 10DAYHERSHBERGER TYPEASSAYIN CASTRATED MALE RAT: PLUS T SEMINALVESICLEWEIGHT, WITH AND WITHOUT FLUID + ¯  ? +? ¯  +? +

VENTRAL PROSTATE WEIGHT + ¯  ? +? ¯ + + +? +

LEVATOR ANI PLUS BULBOCAVERNOSUS WEIGHT + ¯  ? +? ¯  +? +

GROWTH   +  +?

NONREPRODUCTIVE ORGAN WEIGHTS LIVER     LIVER  LIV  EDSTAC Final Report Chapter Five Appendices August 1998 3 P  " Weight of Evidence" Determination: Hold, Go to T2T, or Other T2T T2T T2T Repeat T1S T2T T2T T2T PART 2. Known or expected responses of well characterized toxicants in the EDSTP T1S batttery. The list of endpoin All of the described examples would have sufficient positive responses to be triggered for T2T.

T1S ASSAY ENDPOINT IN VITRO HTPS OR BENCH ASSAYS Steroidoge ne sis Fungicide Thyroid Hormone Analogue Fungicide Woodderived Estrogen Estrogenic Pesticide Antithyroi dDrug HTPS ER/ MVLN ER AGONIST    + +

ABOVE PLUS E2 ER ANTAGONIST

HTPS ER PLUS METABOLISM METAB IS ER AGONIST

ABOVE PLUS E2 METAB IS ER ANTAGONIST

HTPS AR/ CV 1 AR AGONIST

ABOVE PLUS DHT AR ANTAGONIST

HTPS AR PLUS METABOLISM METAB IS AR AGONIST

ABOVE PLUS DHT METAB IS AR ANTAGONIST

HTPS TR TR AGONIST  +

ABOVE PLUS T3 TR ANTAGONIST

HTPS TR PLUS METABOLISM METAB IS TR AGONIST

ABOVE PLUS T3 METAB IS TR ANTAGONIST

IN VITRO TESTIS CULTURE INHIBITION OF P4 OR T SYNTHESIS ++  ? +??

IN VIVO ASSAYS 3 DAY UTEROTROPHIC IN ADULT OVX'D RAT UTERINE WEIGHT WITH AND WITHOUT FLUID     +

UTERINE HISTOLOGY     +

BODY WEIGHT

VAGINAL SMEAR

3 DAY UTEROTROPHIC IN ADULT OVX'D RAT PLUS E2 UTERINE WEIGHT WITH AND WITHOUT FLUID

UTERINE HISTOLOGY

BODY WEIGHT

VAGINAL SMEAR       EDSTAC Final Report Chapter Five Appendices August 1998 4 P  PUBERTAL/ THYROID ASSAY IN WEANLING FEMALE RAT GROWTH  ?    +?

AGE AT VAGINAL OPENING +   ? +  + ?  + +?

WEIGHT AT VAGINAL OPENING +   ? +  + ?  +

SERUM T4  + ¯ ?    ++

SERUM TSH  + ¯ ?    ++

UTERINE WEIGHT WITH AND WITHOUT FLUID +? ¯  ? +? ¯   +?

OVARIAN WEIGHT +   ? +    +?

THYROID HISTOLOGY  +? ¯

NONREPRODUCTIVE ORGAN WEIGHTS LIVER ?   ? LIVER  LIVER  ? LIVER  THYROID  7 10DAYHERSHBERGER TYPEASSAYIN CASTRATED MALE RAT SEMINALVESICLEWEIGHT, WITH AND WITHOUT FLUID

VENTRAL PROSTATE WEIGHT

LEVATOR ANI PLUS BULBOCAVERNOSUS WEIGHT

GROWTH  ?    +?

NONREPRODUCTIVE ORGAN WEIGHTS LIVER ?   +?   LIVER  + ?

7 10DAYHERSHBERGER TYPEASSAYIN CASTRATED MALE RAT: PLUS T SEMINALVESICLEWEIGHT, WITH AND WITHOUT FLUID     + +?

VENTRAL PROSTATE WEIGHT     + +?

LEVATOR ANI PLUS BULBOCAVERNOSUS WEIGHT     +? +?

GROWTH  ?    +?

NONREPRODUCTIVE ORGAN WEIGHTS LIVER ?   LIVER   LIVER  + ?

" Weight  of Evidence" Determination: Hold, Go to T2T, or Other T2T REPEAT T1S, T2T T2T MAY REPEAT T1S, T2T T2T T2T EDSTAC Final Report Chapter Five Appendices August 1998 5 P  PART 3. Known or expected responses of well characterized toxicants in the EDSTP T1S batttery. The list of endpoin T1S ASSAY ENDPOINT IN VITRO HTPS OR BENCH ASSAYS Neurotoxic Pesticide Plasticizer Antiandrogeni cFungicide Antiestrogenic Drug Herbicide Xenoestroge n HTPS ER/ MVLN ER AGONIST + +    ++

ABOVE PLUS E2 ER ANTAGONIST    +

HTPS ER PLUS METABOLISM METAB IS ER AGONIST    +

ABOVE PLUS E2 METAB IS ER ANTAGONIST    +++

HTPS AR/ CV 1 AR AGONIST

ABOVE PLUS DHT AR ANTAGONIST      ?

HTPS AR PLUS METABOLISM METAB IS AR AGONIST

ABOVE PLUS DHT METAB IS AR ANTAGONIST   ++ ?

HTPS TR TR AGONIST

ABOVE PLUS T3 TR ANTAGONIST

HTPS TR PLUS METABOLISM METAB IS TR AGONIST

ABOVE PLUS T3 METAB IS TR ANTAGONIST

IN VITRO TESTIS CULTURE INHIBITION OF P4 OR T SYNTHESIS  ?

IN VIVO ASSAYS 3 DAY UTEROTROPHIC IN ADULT OVX'D RAT UTERINE WEIGHT WITH AND WITHOUT FLUID    +  ++

UTERINE HISTOLOGY    +  ++

BODY WEIGHT +

VAGINAL CORNIFICATION    +  +

3 DAY UTEROTROPHIC IN ADULT OVX'D RAT PLUS E2 UTERINE WEIGHT WITH AND WITHOUT FLUID    ++

UTERINE HISTOLOGY    ++

BODY WEIGHT +

VAGINAL CORNIFICATION    ++   EDSTAC Final Report Chapter Five Appendices August 1998 6 P  PUBERTAL/ THYROID ASSAY IN WEANLING FEMALE RAT GROWTH +

AGE AT VAGINAL OPENING    +? + ¯ ?

WEIGHT AT VAGINAL OPENING    +? + ¯ ?

SERUM T4

SERUM TSH

UTERINE WEIGHT WITH AND WITHOUT FLUID    +

OVARIAN WEIGHT    +

THYROID HISTOLOGY

NONREPRODUCTIVE ORGAN WEIGHTS +

7 10DAYHERSHBERGER TYPEASSAYIN CASTRATED MALE RAT SEMINALVESICLEWEIGHT, WITH AND WITHOUT FLUID

VENTRAL PROSTATE WEIGHT

LEVATOR ANI PLUS BULBOCAVERNOSUS WEIGHT

GROWTH +

NONREPRODUCTIVE ORGAN WEIGHTS +

7 10DAYHERSHBERGER TYPEASSAYIN CASTRATED MALE RAT: PLUS T SEMINALVESICLEWEIGHT, WITH AND WITHOUT FLUID  +? ++

VENTRAL PROSTATE WEIGHT  +? ++

LEVATOR ANI PLUS BULBOCAVERNOSUS WEIGHT  +? +

GROWTH +

NONREPRODUCTIVE ORGAN WEIGHTS +

" Weight of Evidence" Determination: Hold, Go to T2T, or Other REPEAT T1S, Hold1 T2T T2T T2T REPEAT T1S, T2T Other 2 [ Footnote: ( 1) Ofthedescribedexamples, theneurotoxicpesticidewouldgotothe" holdbox" orbestudiedfurtherbeforeitwouldgotoT2T. Footnote ( 2) Thexenoestrogenisonlyestrogenic invitro andbyinjection, butwasnotestrogenicbyoraltreatment. Theuterotrophicassayshouldberepeated with oral dosing to determine if the xenoestrogen was estrogenic by the oral route before it went to T2T.] EDSTAC Final Report Chapter Five Appendices August 1998 Appendix Q Study Designs and Protocols for Recommended Tests in Tier 2 Testing Battery Table of Contents I. Two Generation Mammalian Reproductive Toxicity Study Design . . . . . . . . . . . . . . . . . 1 II. Study Design for the Alternative Mammalian Reproduction Test . . . . . . . . . . . . . . . . . . 3 III. Study Design for the One Generation Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 IV. Recommended Extensions to the Avian Reproduction Guideline . . . . . . . . . . . . . . . . . . 8 V. Fish Life Cycle Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 A. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 B. Materials, Methods, and Reporting Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 C. Literature Cited . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 EDSTAC Final Report Chapter Five Appendices August 1998 1 Q  I. Two Generation Mammalian Reproductive Toxicity Study Design 1 2 The two generation reproductive toxicity study in rats ( TSCA 799.9380, August 15, 1997; 3 OPPTS 870.3800; Public Draft, February 1996; OECD no. 416 1983; FIFRA Subdivision F 4 Guidelines  83 4) is designed to comprehensively evaluate the effects of a chemical on gonadal 5 function, estrous cycles, mating behavior, fertilization, implantation, pregnancy, parturition, 6 lactation, weaning, and the offsprings' ability to achieve adulthood and successfully reproduce, 7 through two generations, one litter per generation. Administration is usually oral ( dosed feed, 8 dosed water, or gavage) but other routes are acceptable with justification ( e. g., inhalation). In 9 addition, the study also provides information about neonatal survival, growth, development, and 10 preliminary data on possible teratogenesis. The experimental design for a two generation 11 reproductive toxicity study is presented in Figure Q 1. 12 13 In the existing two generation reproductive toxicity test, a minimum of three treatment levels and 14 a concurrent control group are required. At least 20 males and sufficient females to produce 20 15 pregnant females must be used in each group as prescribed in this current guideline. The highest 16 dose must induce toxicity but not to exceed 10% mortality. In this study, potential hormonal 17 effects can be detected through behavioral changes, ability to become pregnant, duration of 18 gestation, signs of difficult or prolonged parturition, apparent sex ratio ( as ascertained by 19 anogenital distances) of the offspring, feminization or masculinization of offspring, number of 20 pups, stillbirths, gross pathology and histopathology of the vagina, uterus, ovaries, testis, 21 epididymis, seminal vesicles, prostate, and any other identified target organs. Table 5.3 ( Chapter 22 Five, Section VI, B, provides a summary of the endpoints that are evaluated within the framework 23 of the experimental design of the updated two generation reproductive toxicity test ( and some 24 additions, still under consideration, to cover Estrogen, Androgen, and Thyroid concerns). 25 26 These observations are comprehensive and cover every phase of reproduction and development. 27 Tests that measure only a single dimension or component of hormonal activity, ( e. g., in vitro or 28 short term assays) provide supplementary and/ or mechanistic information, but cannot provide the 29 breadth of information listed in Table 5.3, which is critical for risk assessment. 30 31 Additionally, in this study type, hormonally induced effects such as abortion, resorption, or 32 premature delivery as well as abnormalities and anomalies such as masculinization of the female 33 offspring or feminization of male offspring can be detected. Substances such as the 34 phytoestrogen, coumesterol, and the anti androgen, cyproterone acetate, which possess the 35 potential to alter normal sexual differentiation, were similarly detected in this study test system 36 ( i. e., 1982 Guideline). The initial prebreed exposure period ( ten weeks) of the two generation 37 reproductive toxicity test also provides information on subchronic exposures which can be used 38 for other regulatory purposes. 39 40 EDSTAC Final Report Chapter Five Appendices August 1998 2 Q  Figure Q. 1 1 2 Two Generation Reproductive Toxicity Study Design 3 4 5 PBE M G L FO 6 Q N2 7 N1 8 ECE 9 M G L W PBE M G L 10 F1 N2 11 N3 | N1 12 C VP EC E 13 PPS 14 M G L W 15 F2 N3 16 17 C KEY 18 Q = Quarantine ( one week) 19 PBE = Pre Breed Exposure ( ten weeks) 20 M = Mating ( two weeks) 21 G = Gestation ( three weeks) 22 L = Lactation ( three weeks) 23 VP = Vaginal patency ( evaluated in F1 females on postnatal day 22 to acquisition) 24 PPS = Preputial separation ( evaluated in F1 males on postnatal day 35 to acquisition) 25 W = Weaning ( postnatal day 21) 26 N1 = Necropsy of all paternal animals 27 N2 = Necropsy of all maternal animals 28 N3 = Necropsy of selected weanlings, three/ sex/ litter, if possible 29 ECE = Estrous Cyclicity Evaluations ( three weeks) 30 C = Cull litters to ten pups ( with equal sex ratio) on postnatal day four 31 32 33 Direct exposure via diet, drinking water, inhalation, etc.

34 Possible indirect exposure from transplacental and/ or translactational exposure 35 Both direct and possible indirect exposure if in feed or water ( nursing pups also self feeding and drinking) 36 37 EDSTAC Final Report Chapter Five Appendices August 1998 3 Q  II. Study Design for the Alternative Mammalian Reproduction Test 1 2 Female P0 rats are quarantined for one week prior to mating. Sufficient numbers of animals are 3 used in order to attain 20 pregnant dams per dosage group. During a two week mating period 4 females are removed from breeding cages when sperm positive smears are detected and they are 5 randomly assigned to treatment groups. Chemicals are administered to pregnant female rats by 6 oral gavage beginning on day seven of gestation ( day one being the day sperm positive) and 7 continued throughout gestation and lactation, up to weaning of the F1 generation. F1 pups are 8 never exposed directly, but receive the chemical via transplacental and lactational exposures. 9 10 On the day of birth ( postnatal day ( PND) 0), anogenital distance ( AGD) is measured in both male 11 and female F1 pups. Animals with ambiguous gender should be uniquely identified such that sex 12 can be identified at a later age. At about four days of age, litters should be standardized to eight 13 pups ( four males and four females where possible) by randomly removing excess pups ( necropsy 14 to confirm sex). At about twelve days of age, the pups are examined for the presence of 15 nipples/ aerolas, which should be clearly visible in female but not male pups. ( These endpoints are 16 sensitive to androgens and antiandrogens.) 17 18 After weaning at 21 days of age, pups ( at least 20/ sex/ dose) are housed in unisexual pairs until 19 mating ten weeks later to produce the F2. All surplus weanlings are necropsied and examined 20 externally and internally for reproductive tract malformations. P0 parental females will be 21 necropsied at the weaning of their F1 litters on PND 21. These dams will be subjected to a gross 22 necropsy with organ weights: thyroid, ovaries, pituitary, uterus ( count implants), kidneys, and 23 liver. 24 25 After weaning, female F1 retained rats are examined daily, beginning on PND 22, for vaginal 26 opening until complete. Both age and weight at vaginal opening are determined. For retained F1 27 male pups, the age of preputial separation is determined from PND 35 on, with age and weight at 28 preputial separation recorded. 29 30 Three weeks prior to mating of the F1, estrous cyclicity is monitored for three weeks in female 31 rats. Following this, F1 non littermate pairs of males and females from the same dose group are 32 established for a two week period in order to produce the F2 generation. Upon delivery, F2 pups 33 are counted by sex and weighed, being terminated at weaning after individual body weights are 34 taken. The F2 litters are not standardized to eight pups per litter. 35 36 After mating the F1 males are necropsied and reproductive organs ( SV, VP, LA, testis, 37 epididymides, pituitary, etc.) are weighed and preserved for histological examinations. In 38 addition, one testis and caput/ corpus and cauda epididymis is used for enumeration of sperm 39 numbers. The thyroid gland also is weighed and saved for histological examination and serum is 40 taken for determination of serum T4, T3 and TSH ( especially if testing was triggered by thyroid 41 endpoints in screening). Liver, kidney, and brain weights also should be determined. 42 43 EDSTAC Final Report Chapter Five Appendices August 1998 4 Q  F1 females should be necropsied immediately after weaning of the F2 pups and reproductive 1 organs ( ovary, uterus, pituitary) weighed and saved for histopathological examination. Liver, 2 kidney and brain weights also should be taken. The thyroid gland also should be weighed and 3 saved. 4 5 III. Study Design for the One Generation Test 6 7 A. Rats of both sexes seven nine weeks of age, quarantined for one week, distributed into 8 treatment groups ( four is usual, can use any number depending on previous information) 9 ten/ sex/ group, then exposed separately ( housed individually) to chemical substance or mixture 10 by whatever route ( we have used gavage, IV injection, inhalation, dosed feed, dosed water) 11 for a prebreed exposure period ( suggest two weeks but would depend on any prior 12 information on chemical substance or mixture in terms of bioaccumulation, cumulative 13 toxicity, etc.; could delete to shorten duration) 14 B. Animals mated one: one within groups for two weeks ( can use one week to shorten 15 duration); vaginal sperm/ copulation plug indicative of insemination with date designated 16 gestational day ( gd) 0. On gd 0, females (" dams") separated from males and housed 17 individually. 18 C. Dams deliver on gd 22 ± one ( for rats), date of delivery designated postnatal day ( pnd) 0. 19 Anogenital distance measured in newborn pups ( pnd 0 or one). Litters ( designated F1) culled 20 to standard size on pnd four ( usually eight ten/ litter, with as even a sex ratio as possible). 21 Culled pups could be examined, or discarded. F0 males necropsied after all dams deliver, with 22 organ weights, blood samples (?), andrological assessments (?). 23 D. At weaning on pnd 21, F0 dams necropsied, with organ weights, blood samples (?), etc., F1 24 one three pups/ sex/ litter necropsied with organ weights, blood sampling (?). 25 E. Also at weaning, pups selected from all litters, ten/ sex/ group, to undergo postwean exposure. 26 F. During ten week post wean exposure period, F1 females assessed for vaginal patency ( VP; 27 starting on pnd 22), F1 males assessed for preputial separation ( preputial separation; starting 28 on pnd 35). For the last three weeks, F1 females assessed for vaginal cyclicity by daily vaginal 29 smears ( can terminate after VP and preputial separation). 30 G. On pnd 91, F1 males and females necropsied with organ weights, blood sampling, 31 andrological assessments, etc. 32 33 Final ESDTAC Report Chapter Five Appendices August 1998 Q  5 Figure Q. 2: Alternative Mammalian Reproductive Test ( AMRT) 1 2 0 31 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 4 5 G 67 P F1 10 week holding period 8M L 20/ sex/ group M G L Q 9 10 11 W gd 0 pnd 0 pnd 21 12 gd 0 gd 6 pnd C VP EC 13 21 PPS 14 15 pnd 0 pnd 12 16 AGD nipples N F1 males N F1 Females 17 Organ weights Organ weights 18 N F0 Females Histology Histology 19 Gross necropsy Sperm T3/ T4 ® TSH 20 Organ weights T3/ T4 ® TSH 21 ( thyroid, ovaries, pituitary, uterus, liver, kidneys, brain) F2 Weanlings Count, sex, weigh,

22 discard 23 N Subset F1 Weanlings 24 Gross necropsy ( reproductive tract malformations) 25 KEY: 26 Dose F0 females Q = Quarantine M = Mating ( 2 weeks) G = Gestation P = Parturition L = Lactation VP = Vaginal Patency PPS = Preputial Separation EC = Estrous Cyclicity gd = Gestational Day pnd = Postnatal Day AGD= Anogenital Distance Final ESDTAC Report Chapter Five Appendices August 1998 Q  5 N = Necropsy C = Cull F1 pups on pnd 4 Final EDSTAC Report Chapter Five Appendices August 1998 6 Q  H. Information on: 1 · General toxicity ( body weights, feed and/ or water consumption, chemical substance or mixture 2 intake, clinical observations, etc.) 3 · Mating 4 · Fecundity 5 · Fertility 6 · Parturition ( number total, dead, live, sex, weight, AGD, gross exam) 7 · Prenatal survival and growth 8 · Postnatal survival and growth 9 · Reproductive development ( VP, preputial separation) 10 · Vaginal cyclicity in F1 females 11 · Male epididymal sperm number, motility, morphology; testicular homogenization resistant 12 spermatid head counts ( SHC), daily sperm production ( DSP), efficiency of DSP 13 · Blood samples for E, A, T ( T4, TSH) 14 · Organ weights: reproductive organs, thyroid, liver ( EROD?) 15 · Histology of reproductive organs, thyroid, brain, etc., optional 16 I. Advantages include: 17 · Involves in utero exposure 18 · Assesses reproductive development, structures and functions. 19 · Can be modified as needed, e. g., shorten/ lengthen/ delete prebred exposure, shorten mating, shorten 20 postwean exposure ( to only through reproductive development or to ~ pnd 50), after postwean 21 exposure, mate F1 to produce F2 generation ( terminate F2 on pnd four, etc.), add other assessments 22 ( e. g., more blood samples, histopathology, etc.), add F1 developmental landmarks such as surface 23 righting reflex, pinna detachment, eye opening, acquisition of startle reflex, midair righting reflex, 24 motor activity, learning and memory, etc. 25 · Covers the mammalian " waterfront" for E, A, and T. 26 · Any contract lab currently performing multi generation studies has the necessary capabilities ( staff, 27 facilities, SOPs, experience). This is relatively " low tech" except for the blood assays and 28 andrological assessments. 29 J. Sensitivity: 30 The sensitivity is dependent on the number of animals/ group ( for statistical power), the type of 31 assessments included, the number of treatment groups and the range of doses. It is a powerful, apical 32 assay. 33 34 Final EDSTAC Report Chapter Five Appendices August 1998 7 Q  Figure Q. 3 1 One Generation Mammalian Reproductive Toxicity Test 2 3 4 PBE M G L FO 5 Q 6 2 wks 2 wks 3 wks 3 wks N, B, O 7 F0 females 89 N, B, A, O 10 F0 males 11 12 G P L W 10 week PWE

13 91 d 14 15 16 Cull to N VP VC 17 8 10/ Litter 1 3/ sex/ Litter PPS 18 KEY: 19 N, B, A, O Q20 = Quarantine AGD F1 males PBE21 = Prebred exposure and females M22 = Mating G23 = Gestation L24 = Lactation N25 = Necropsy B26 = Blood samples A27 = Andrological assessments O28 = Organ weights P29 = Parturition ( delivery, pnd 0)

AGD30 = Anogenital distance VP31 = Vaginal patency PPS32 = Preputial separation VC33 = Vaginal cyclicity PWE34 = Postwean exposure 35 Direct exposure 36 Possible indirect transplacental and/ or translactational exposure 37 Direct and possible indirect exposure if route is dosed feed or water 38 39 Final EDSTAC Report Chapter Five Appendices August 1998 1 Q  IV. Recommended Extensions to the Avian Reproduction Guideline A. The following modifications of OPPTS 850.2300 are recommended to assess development in the offspring of exposed adults.

· modify ( d) Test procedures ( 2) Definitive test, ( viii) Chick ..., to require that all offspring be maintained separately by parental ( dose) group; and,

· modify ( d) Test procedures ( 2) Definitive test, ( ix) Observations of record on chicks... to include:

1. the genetic sex ratio at hatching; genetic sex can be determined routinely by the identification of restriction fragments from sex chromosomes derived from red blood cells in a small blood sample (~ 0.3ml) ( Halvorsen 1990; Dvorak et al. 1992); identification of the genetic sex of offspring allows closer control of subsequent operations, as well as providing an endpoint of interest.

2. at 14 days of age, for 12 genetic males at each dose level:

· external characteristics, the size of cloacal protuberance in ducks and cloacal gland size in quail ( Ottinger, M. A. and H. J. Brinkley., other refs); and,

· internal characteristics, the size and dimorphism of gonads, the presence of oviducts, especially on the left side, and the histology of the gonad, looking especially for the relative amount of cortex and the presence of primary germ cells located there, ( Fry et al., 1987; Ottinger and Bakst,

1981); also, serum sex steroid ( estrogens, androgens) should be determined ( more refs needed)

3. at 14 days of age, for 12 genetic females at each dose level:

· internal measurements only, looking especially for presence of structures on the right side, and histologically the relative amount of cortex and medulla and the development of oocytes; also,

serum sex steroid ( estrogens, androgens) should be determined.

4. at 14 days of age, both sexes at each dose level will be assessed for general growth parameters indicative of adequate thyroid function major organ weights, including brain, body weights, wing and bone length, thyroid weight, and skeletal x ray. If there are significant differences among exposure groups in these responses, thyroid histopathology will be performed on all groups. Otherwise, only the high dose and control groups will be so assessed. Final EDSTAC Report Chapter Five Appendices August 1998 2 Q  5. at 14 days of age all surviving chicks will be subjected to the visual cliff test ( Baxter et al., 1969;

Dahlgren and Linder, 1971; Emlen, 1963; Fleming et al., 1985; Fox, 1976). The F1 chicks will be challenged with a cold stress test ( Fleming et al., 1985a; Fleming et al., 1985b; Maguire and Williams, 1987; Martin and Solomon, 1991; Rattner et al., 1982; Rattner et al., 1987; Tori and Mayer, 1981) and the nest attentiveness test ( Fox et al., 1978; McArthur et al., 1983; Kubiak et al.,

1989). Final EDSTAC Report Chapter Five Appendices August 1998 3 Q  · Breeding the Second Generation:

Complete assessment of the potential endocrine disruption of chemical substances or mixtures requires a test to the reproductive capability of birds exposed as embryos. This is distinct from data gathered in part A that evaluates the progress of sexual differentiation and reproductive development in birds exposed as embryos. There are no codified guidelines for a two generation avian reproduction test, however, an OECD work group is currently designing protocols for such a test.

These guidelines should be used if they will be available soon enough to implement in Tier 2 Testing.

If not, development of a protocol by the EPA should be a very high priority.

A two generation avian reproduction test should have the following features:

· uses subjects that mature and breed in a short time; Japanese quail are an obvious choice, and in addition there is substantial information on the husbandry, development, endocrinology and reproduction of this species,

· exposure of the parental generation should be exposed through the feed before and continue through egg laying; chicks ( F1 generation) should be fed from hatching diets at the same dose levels as P1,

· breeding of F1 should begin as soon as possible, consistent with the biology of the species used;

Japanese quail mature in about eight weeks, and should be kept on long day photoperiod from hatching,

· responses measured should follow the guidelines for the Avian Reproduction test ( OPPTS 850.2300) with the addition of measurement of the age at reproduction in both sexes.

· From the 12 hens per treatment group, 60 eggs will be needed with 30 eggs or survivors thereof sacrificed at 14 days and 30 eggs/ chicks maintained for F1 testing.

An efficient method for conducting a two generation test would be to use the Japanese quail in a modified Avian Reproduction test as described in part A, and produce extra offspring that would the F1 breeders in a two generation test. Final EDSTAC Report Chapter Five Appendices August 1998 4 Q  B. In the expanded two generation testing protocol described in section A above, exposure of F1 embryos occurs only through maternal depositions of chemical substance or mixtures to eggs. Often avian egg exposure occurs directly, such as during spraying of pesticides and pesticide fertilizer mixtures or crude oils and derivatives during spills, or with certain air pollutants. Direct exposure differs from maternal deposition because the parent compound is not being introduced to the egg as adults metabolize the parent compounds. These facts argue for an alternative exposure to embryos via the direct pathway. For chemicals that have pathways directly to eggs, ( certain pesticides, fertilizers, oils,

and some air pollutants), a direct exposure protocol needs to further amend existing EPA method ( OPPTS 850.2300). Experience with this assessment protocol is well documented ( Hoffman and Eastin, 1981; Hoffman and Albers, 1984; Hoffman, 1990; Somers et al., 1974). It is recommended that eggs be dipped in appropriate solutions, including registered carrier solvents for pesticides for ten seconds at 84 hrs of incubation for bobwhite quail and mallards and 63 hours for Japanese quail. This assures embryonic development is proceeding uniformly prior to exposure ( eggs can be candled) and during the period of organogenesis. Eggs will be air dried for five minutes prior to return to the incubator. All eggs will be clean eggs with no previous adult exposures. Surplus control group eggs or purchased eggs may be used. Testing of these eggs and offspring will be identical to those prescribed in Section A, 1, 5 above.

· Functional Endpoint Tests for the Two Generation Avian Reproduction Test:

i. Nest Attentiveness/ Incubation Behavior: Measuring nest attentiveness assessment of adult birds during incubation would complement other proposed toxicity endpoints. It is a functional test of adult reproduction behavior following embryonic and subsequent dietary exposure and can provide a functional endpoint of high ecological relevance.

ii. Visual Cliff Test: A short term behavioral assessment of hatchling chicks to a visual challenge. It evaluates depth perception and motivation needed for escape responses and predatory performance to capture live prey. A functional deficit in these behavior patterns early in life can result in reduced growth and survival, both of which are ecologically important. Final EDSTAC Report Chapter Five Appendices August 1998 5 Q  iii. Cold Stress Test: The ability of chicks and adult birds to regulate body temperature is a vital physiological response and largely controlled by thyroid hormones. Delay or impairment of thermoregulatory ability might significantly affect survival during inclement weather.

Likewise, adult birds are exposed to winter cold stress and inclement weather during migration periods. This test provides a functional evaluation of the effect of cold stress on body temperature and the ability to regulate body temperature within normal ranges.

V. Fish Life Cycle Test A. Introduction 1. Purpose The fish life cycle test is designed to evaluate growth, maturation, and reproduction of fish exposed to a chemical, substance, or mixture from a continuous exposure through a complete reproductive life cycle.

2. Test Material The test material must be soluble or dispersable to allow distribution to test aquaria.

3. Acceptable Protocols Test protocols which provide guidance for performing a fish life cycle test can be found in the following references.

Benoit, D. A., " User's Guide for Conducting Life Cycle Chronic Toxicity Tests with Fathead Minnows ( Pimephales promelas)," Environ. Res. Lab.  Duluth, MN. EPA 600/ 8 81 011, 1981 Final EDSTAC Report Chapter Five Appendices August 1998 6 Q  Hansen, D. J., P. R. Parrish, S. C. Schimmel, and L. R. Goodman, " Toxicity Test Using Sheepshead Minnows ( Cyprinodon variegatus)," Bioassay Procedures for the Ocean Disposal Permit Program, EPA 600/ 9: 78 010, 1978.

American Public Health Association, American Water Works Association and Water Pollution Control Federation, " Standard Methods for the Examination of Water and Wastewater," 16: 854,

1985.

B. Materials, Methods, and Reporting Requirements [ Note: numbers in parentheses refer to the numbered references at the end of this section.]

1. Biological System a) Preferred Species The preferred test species are fathead minnow ( Pimephales promelas) and sheepshead minnow ( Cyprinodon variegatus).

b) Source and Acclimation of Fish Adult fish are obtained from either wild populations or suitable culture laboratories. Fathead minnows should be maintained at 25o C. Sheepshead minnows are to be held in flowing 30o C seawater of > 15 o/ oo salinity. Source fish should be held for at least two weeks prior to breeding and should not exhibit mortalities > 5 %.

c) Eggs from Adult Fish Artificial inducement and natural spawning are the two methods for obtaining a sufficient number of eggs for a chronic exposure. Artificial inducement entails the stimulation of egg production by injection of human gonadotrophic hormone. Sheepshead minnow females can be injected intraperitoneally with five IU HCG on two consecutive days. Two days following the second injection, ova from females are stripped and mixed with sperm derived from excised macerated testes. Usually ten females and five males should be used.( l) Final EDSTAC Report Chapter Five Appendices August 1998 7 Q  Natural spawning is possible with a few considerations for each fish species and is preferred.

Fathead minnows require paired spawning in order to eliminate fighting and competition.

Culture units for this fish can consist of one tank measuring 30.5 x 30.5 x 61 cm with a water depth of 18 cm and four individual spawning chambers ( 15.2 x 30.5 cm) formed by stainless steel screen dividers ( 5 mesh, 0.89 mm wire).( 2) Sheepshead minnow embryos are obtained by combining five or more females and three males in spawning chambers measuring 20 x 35 x 22 cm. Mature adults should attain a minimum standard length of 2 > mm and display courtship characteristics ( sexual dimorphism, territoriality, and aggressive behavior by the male). Fish from each spawning group are left in chambers for a minimum of 14 days.( 2)

Adult deaths during spawning should be noted; dead animals are removed, but not replaced. At termination of each spawning group, lengths and weights of individual fish are measured.

d) Feeding Fry of both fish species should be fed equal portions of live brine shrimp nauplii at least two times daily about six hours apart for three weeks ( frozen nauplii are not to be used). juveniles ( four weeks posthatch) and adults can be fed twice daily on equal portions of dry food ( e. g.,

Tetramin or Biorell) supplemented with frozen adult brine shrimp. Each batch of food should be checked for pesticides and metals.

e) Embryo Removal A record of numbers and egg fertility must be maintained daily. All embryos are examined daily with a dissecting scope or magnifying viewer to remove empty shells and opaque, or abnormal appearing embryos. If less than 50 percent of the embryos from a spawn appear to be healthy and fertile, all embryos from that spawn should be discarded. ( 2) Embryos should be removed at a fixed time each day so spawning activity is not disturbed unnecessarily.

f) Embryo Exposure ( Four Five Days)

The life cycle chronic toxicity test must begin with embryos from at least three separate spawnings that are < 24 hours old and have soaked in dilution water for at least t waugh hours. Final EDSTAC Report Chapter Five Appendices August 1998 8 Q  ( 2) Testing begins by randomly distributing 50 embryos to each of the four replicate larval growth chambers. ( 2) Ten embryos are transferred with a large bore eye dropper to successive incubation cups which are standing in dilution water. This is repeated until 50 embryos are in each cup. The incubation cups are then distributed to each replicate larval chamber.

Survival of embryos, time required to hatch, hatching success, and survival of fry for four weeks are determined and recorded. Dead embryos usually turn opaque and must be counted and removed each day until hatching is complete. Live fungused embryos must be removed daily and counted as dead.( 2)

g) Larval Juvenile Exposure ( Eight Weeks)

After hatching, each group of larvae is randomly reduced to 25, and released in replicate larval growth chambers. ( 2) This random selection must include any fish that are lethargic or deformed.

Survival should be determined in each replicate growth chamber at least once a week. Survival during this period is determined by counting the number of live fish, since dead larvae deteriorate rapidly.

At four and eight weeks after hatching, total lengths ( mm) of all fish must be recorded. ( 2)

Techniques suggested for measuring fish include direct measurement and a photographic method outlined by McKim and Benoit 1971. ( 3) In order to treat growth as a valid endpoint, the amount of food given to the control and treated fish must be kept constant between exposures.

h) Juvenile Adult Exposure ( 32 40 Weeks)

All fish are transferred to the adult spawning tank ( same concentration) eight weeks after hatching. ( 2) Each tank should have 25 randomly selected fish ( deformed individuals included).

When secondary sexual characteristics are well developed, fathead minnow ( 20 24 week post hatch) males will exhibit tubercles, pads and body color, while females will exhibit extended transparent and canals ( urogenital papilla). At this time, mature fish should be placed in spawning tank, separate from undeveloped fish. ( 2) The spawning tank will be divided into four Final EDSTAC Report Chapter Five Appendices August 1998 9 Q  individual spawning chambers with appropriate spawning substrates. Four males and four females are randomly chosen and assigned to spawning chambers. Substrates are examined daily and embryos removed, counted, and recorded separately for each pair.

The adult exposure ( fathead minnow) should be terminated when, during the decreasing daylength photoperiod, a one week period passes in which no spawning occurs. ( 2) Testing using sheepshead minnows should terminate after spawning is observed for two weeks because this fish spawns readily and almost daily unless immature or affected by a pollutant. ( l)

i) Second Generation Embryo Exposure ( Four Five Days)

Fifty embryos from each concentration level are randomly selected and transferred to incubation cups for hatch. Those embryos not selected are discarded. Test procedures used during embryo removal and embryo exposure ( sections A, 5 and A, 6, respectively) are repeated with second generation embryo reexposure. ( l, 2)

j) Second Generation Larval juvenile Exposure ( Four Eight Weeks)

Eight week exposure begins with the release of two groups of 25 larvae in replicate growth chambers. These larvae should have been produced from different breeding pairs in each spawning tank. Selection of each group should be from early spawnings. ( 1, 2) Testing procedures are the same as those described in section A, 7.

Each group of second generation fish is terminated eight weeks after hatching. Fish are blotted,

weighed, and measured before being discarded. ( l, 2)

2. Physical System a) Test Water · Fathead Minnow i. Test water can be supplied from a well or spring provided that the source is not polluted; Final EDSTAC Report Chapter Five Appendices August 1998 10 Q  ii. Water should be sterilized with ultra violet irradiation and tested for pesticides, heavy metals, and other possible contaminants;

iii. Hardness of 40 to 48 mg/ L as CaCO3 and pH of 7.2 to 7.6 is recommended;

iv. Reconstituted water can be used. Detailed descriptions of acceptable procedures for preparing diluent are found in the protocols by the American Society of Testing Materials 1980. ( 4)

· Sheepshead Minnow i. Test water may be natural ( sterilized and filtered to remove particles 15 microns and larger) or a commercial mixture ( provided that there are no adverse affects to test organisms or alterations in test material toxicity);

ii. Natural seawater is considered to be of constant quality if the weekly range of salinity is less than six percent, and if monthly pH range is less than 0.8 of a pH unit;

iii. Salinity should be > 15 parts per thousand;

iv. Water must be sterilized and free of Pollutants. ( l) Irradiation with ultraviolet light is recommended to Sterilize test water.

b) Temperature A continuous record of temperature of test water must be kept.

· Fathead Minnow Temperature should be maintained at 25 ° C and should not remain outside the range of 24 to 26 ° C for more than 48 hours. ( 2)

· Sheepshead Minnow Temperature should be maintained at 30 ° C. ( l)

c) Photoperiod Lighting above each replicate must be balanced and must simulate the wavelength spectra of sunlight. Light intensities at the water surface should range from 10 to 100 lumens. One lumen per square meter is equal to one lux.

· Fathead Minnow A graduated photoperiod as described in Benoit ( 1981) is used. Final EDSTAC Report Chapter Five Appendices August 1998 11 Q  · Sheepshead Minnow A 16 hour light/ 8 hour dark cycle is maintained throughout the test. ( l)

d) Dosing Apparatus Intermittent flow proportional diluters as described by Mount and Brungs ( 5) or continuous flow serial diluters, as described by Garton ( 6) should be employed. A minimum of five toxicant concentrations with a dilution factor not greater than 0.50 and one control should be used.

e) Toxicant Mixing A mixing chamber is recommended to assure adequate mixing of test material. Aeration should not be used for mixing. Separate flow splitter delivery tubes should run from this container to each replicate larval and adult tank. ( 2) Depending upon the apparatus used a mixing chamber may not be required. T t must, however, be demonstrated that the test solution is completely mixed before introduction into the test system. Flow splitting accuracy must be within 10 percent and should be checked Periodically for accurate distribution of test water to each tank.

( 2)

f) Test Tanks All test tanks should be of either all glass or glass with a plastic or stainless steel frame.

· Fathead Minnow Adult spawning tanks should measure 30.5 x 30.5 x 91.4 cm or 30.5 x 30.5 x 61 cm long with a screened off or separate larval tank. ( 2) Each larval section is divided in half allowing for two larval growth chambers for each adult spawning tank. Larval chambers should be designed with glass bottoms and drains that allow water to be drawn down to 3 cm. ( 2) Test water must be delivered separately to each adult tank and larval section, with one third of the water volume going to the latter. Larval tanks can also be conveniently located directly above spawning tanks containing test solutions of the same concentrations so they can be drained directly into the spawning tank. Test water depth in adult tanks and larval chambers should be a minimum of 15 cm. ( 2)

· Sheepshead Minnow Final EDSTAC Report Chapter Five Appendices August 1998 12 Q  Tanks 45 x 90 x 26 cm with a water depth of 19 cm have been successful. Larval chamber design and test water divided are the same as described for fathead minnow. ( l)

g) Embryo and Fry Chambers Embryo incubation chambers should be made from 120 ml glass jars with the bottoms replaced with 40 mesh stainless steel or nylon screen. Chambers can be oscillated vertically ( 2.5 to 4.0 cm) in the test water ( rocker arm apparatus, 2 rpm motor) or placed in separate chambers with self starting siphons. Both methods should insure adequate exchange of water and test material.

( 1, 2)

h) Flow Rate Flow rates to adult tanks or larval chambers should provide 90 percent replacement in 8 to 12 hours. ( 2) Flow rate must be capable of maintaining dissolved oxygen at above 75 percent of saturation and maintain the toxicant level ( concentration cannot drop below 20% with fish in the tank).

i) Aeration Dilution water should be aerated vigorously insuring that dissolved oxygen concentration will be at or near 90 to 100 percent saturation. Test tanks and embryo chambers should not be aerated.

( l, 2)

3. Chemical System a) Concentrations A minimum of five concentrations of toxicant and a control ( all duplicated) are used in this chronic test. A solvent control is added if a solvent is utilized. As a minimum, the concentration of toxicant must be measured in one tank at each toxicant level every week. Water samples should be taken about midway between top and bottom and the sides of the tank. Final EDSTAC Report Chapter Five Appendices August 1998 13 Q  One concentration selected must adversely affect a life stage and one concentration must not affect any life stage. ( 1)

b) Measurement of other Variables Dissolved oxygen must be measured at each concentration at least once a week. Freshwater parameters in a control and one concentration must be analyzed once a week. These parameters should include pH, alkalinity, hardness, and conductance. Natural seawater must maintain a constant salinity and not fluctuate more than six percent weekly or a monthly pH range of less than 0.8 of a pH unit. ( 2)

c) Solvents If solvents other than water are necessary, they should be used sparingly and not to exceed 0.1 mL/ L in a flow through system. The following solvents are acceptable: ( 4)

· dimethylformamide · triethylene glycol · methanol · acetone · ethanol The development of chemical saturators for use with hydrophobic chemicals may be used with most test chemicals. ( 7, 8, 9)

d) Calculations Data from these toxicity studies are of two types, continuous ( i. e., length, weight) and discrete ( i. e., number of fish hatching or surviving). In general, continuous data should be analyzed with the appropriate analysis of variance ( ANOVA) technique followed by an appropriate multiple comparison test. Dichotomous data should be analyzed using some form of a 2 x 2 contingency table. Final EDSTAC Report Chapter Five Appendices August 1998 14 Q  As a part of the ANOVA, it is desirable to plot the residuals versus concentration and determine whether there have been any obvious violations of homoscedasticity on the assumption of normality. All test results must be accompanied by the original ( raw) data for the reviewer's evaluation.

C. Literature Cited Hansen, D. J., P. R. Parrish, S. C. Schimmel, and L. R. Goodman, " Toxicity Test Using Sheepshead Minnows ( Cyprinodon variegatus)," Bioassay Procedures for the Ocean Disposal Permit Program, EPA

600/ 9 78 010, 1978.

Benoit, D. A., " User's Guide for Conducting Life Cycle Chronic Toxicity Tests with Fathead Minnows ( Pimephales promelas)," Environ. Res. Lab., Duluth, MN. EPA 600/ 8: 81 011, 1981.

McKim, I. M., D. A. Benoit, " Effect of long term exposures to copper and survival, reproduction and growth of rainbow trout ( Salvelinus fontinalis)," J. Fish. Res. Board Can., 28: 655 662, 1971.

ASTM Standard E 729 80, " Practice for Conducting Acute Toxicity Tests with Fishes,

Macroinvertebrates, and Amphibians," American Society for Testing and Materials, 1916 Race street, Philadelphia, PA 19103, 1971.

Mount, D. I., W. A. Brungs, " A simplified dosinq apparatus for fish toxicology studies," Water Res.,

1: 21 29, 1967.

Garton, R. R., " A simple continuous flow toxicant delivery system," Water Res., 14: 227 230 1980.

Chadwick, G. C., and U. Kiigemagi, " Toxicity evaluation of a technique for introducing dieldrin into water," J. Fish. Res. Board Can., 40: 76 82, 1968.

Gingerich, W. H., W. K. Seim, and R. D. Schonbrod, " An apparatus for the continuous generation of stock solutions of hydrophobic chemicals," Bull. Environ. Contam. Toxicol., 23: 685 689, 1979.

Veith, G. D., and V. M. Comstock, " Apparatus for continuously saturating water with hydrophobic organic chemicals," J. Fish. Res. Board Can., 32: 1849 1851, 1975.

U. S. EPA Pesticide Reregistration Rejection Rate Analysis ( EPA 738 R 94 035), United States Environmental Protection Agency. Washington, DC., 1994. EDSTAC Final Report Chapter Five Appendices August 1998 1 R  Appendix R 1 2 Preliminary Categorization of Tier 1 Screens and Tier 2 Tests 3 by the Screening and Testing Work Group 4 5 As mentioned in Chapter Five, the screens and tests being recommended by the EDSTAC vary 6 considerably in terms of the effort necessary to be fully validated and standardized. During their 7 deliberations, the Screening and Testing Work Group ( STWG) attempted to categorize the levels 8 of validation required of the recommended screens and tests and their preliminary efforts are 9 found in this Appendix. This information is included to give the reader a sense of where the 10 recommended screens and tests may exist across the spectrum of validation; however, the 11 EDSTAC agreed not to try to reach consensus agreement on where all the screens and tests lie, as 12 they will all need to validated and standardized before being included in the Endocrine Disruptors 13 Screening and Testing Program ( EDSTP). The EDSTAC recommends that EPA update this 14 categorization scheme as part of their validation and standardization program. 15 16 The recommended screens and tests ( including all endpoints) will have to meet all the criteria of 17 relevance and reliability for use in regulatory toxicity screening or testing for Estrogen, Androgen, 18 and Thyroid ( EAT) in order to be considered fully validated and standardized ( ICCVAM, 1996; 19 Zeiger, 1998). As screens and tests become fully validated and standardized, they will warrant 20 inclusion in the EDSTP according to their specific and appropriate use. None of the screens, new 21 tests, or enhancements to existing test guidelines included in Tier 1 Screening ( T1S) or Tier 2 22 Testing ( T2T) completely fulfill these criteria to date. As mentioned throughout Chapter Five, 23 each assay and test under consideration in T1S or T2T needs some level of standardization, 24 validation, methods development, or further research before being accepted as a regulatory 25 toxicity screen or test. The level of standardization and validation varies according to a variety of 26 criteria applied to each of the assays, including: period of time in use, existing level of general 27 acceptance in the endocrine toxicology field, and existing understanding of relevancy and 28 reliability. 29 30 The STWG placed the proposed screens and tests in one of four general categories with regard to 31 the level of validation, standardization, or methods development required. A fifth category, 32 discussed in Chapter Five, also identified assays requiring further research. 33 34 Category I: 35 36 Screens and tests which have been fully validated and standardized are placed in Category I. 37 These procedures meet all the criteria of relevance and reliability for use in regulatory toxicity 38 screening or testing for estrogen, androgen, and thyroid. As other procedures become sufficiently 39 standardized and validated to warrant inclusion in Category I, such screens and tests should be 40 incorporated into the EDSTP according to their specific and appropriate use. Only the following 41 tests are included in Category I: 42 43 EDSTAC Final Report Chapter Five Appendices August 1998 2 R  · Two Generation Mammalian Reproductive Toxicity Study ( 1996 Public Draft 1 Guidelines and 1997 TSCA Final Guidelines) 2 · One Generation Test 3 4 Category II: 5 6 Screens and tests which have been in use for a sufficient period of time and which have gained 7 sufficient general acceptance within the field of endocrine toxicology to be considered de facto 8 validated ( reliable and relevant) are included in Category II. These assays measure relevant 9 endpoints, are responsive to endocrine active compounds with a high degree of specificity, are 10 sufficiently sensitive to identify all known active agents, and can reasonably be expected to give 11 reproducible results from laboratory to laboratory, assuming a general level of competence and 12 expertise. Nonetheless, variations in protocols for these screens and tests can produce disparate 13 results. Therefore, standardization of the protocol to be recommended for these screens and tests 14 should be accomplished by EPA before these assays are implemented as screening requirements 15 for endocrine activity or disruption. The following screens and tests are included in Category II: 16 17 · ER Binding Assay 18 · AR Binding Assay 19 · Rodent 3 Day Uterotrophic Assay ( Subcutaneous) 20 · Rodent 5 7 Day Hershberger Assay 21 · Rodent 3 Day Uterotrophic Assay ( Intraperitoneal); 22 · Avian Reproduction Test ( with Bobwhite Quail and Mallard) ( as currently performed) 23 · Fish Life Cycle Test ( Fathead Minnow) Test ( as currently performed) 24 · Mysid Life Cycle Test ( Americamysis) 25 26 Category III: 27 28 Screens and tests which have sufficiently broad use to be generally considered relevant OR 29 reliable to either screening for endocrine activity ( Tier 1) or to testing for adverse endocrine 30 mediated effects ( Tier 2) are included in Category III. These assays cannot, however, be 31 generally considered to be both relevant and reliable. The level of performance that can be 32 expected of these assays with respect to identifying endocrine active agents or endocrine 33 disruptive effects of chemicals must be clarified. Therefore, these assays should undergo further 34 but focused validation and standardization to define their relevance and reliability for the task of 35 endocrine disruptor screening or testing. The validation required may be focused to answer 36 specific questions about relevance and to provide information regarding specificity and sensitivity. 37 The following screens and tests are included in Category III: 38 39 · ER Transcriptional Activation Assay 40 · AR Transcriptional Activation Assay 41 · Steroidogenesis Assay with Minced Testis 42 · Rodent 20 Day Pubertal Female Assay With Thyroid 43 EDSTAC Final Report Chapter Five Appendices August 1998 3 R  · Placental Aromatase Assay 1 · Rodent 14 day Intact Adult Male Assay with Thyroid 2 · Rodent 20 day Thyroid/ Pubertal Male Assay 3 · Alternative Mammalian Reproduction Test 4 · Avian Reproduction Test  when performed in multiple generations 5 · Turtle Egg Assay 6 7 Category IV: 8 9 Screens and tests which may have relevance to the task of either screening for endocrine activity 10 or testing for endocrine disruptive effects, but whose performance in identifying endocrine active 11 agents or endocrine disruptive effects has seen only limiting testing are included in Category IV. 12 Questions as to whether these assays measure endpoints that are relevant to endocrine activity or 13 endocrine disruptive effects, whether these assays respond with specificity and sensitivity to 14 known endocrine active agents, or whether they identify endocrine disruptive effects cannot be 15 addressed with information currently available. In addition, questions regarding the specific 16 protocols and conditions under which the assays should be conducted must be answered before 17 relevance and reliability can be assessed. Nonetheless, these assays would have sufficient utility, if 18 further developed and validated, to enhance or augment the screening and testing program. The 19 following screens and tests are included in Category IV: 20 21 · Frog Metamorphosis Assay 22 · Fish Gonadal Recrudescence Assay 23 · 14 Day ( PND 9 22) Developmental/ Thyroid Assay 24 25 26 EDSTAC Final Report Chapter Five Appendices August 1998 S  1 Appendix S 1 2 Survey of Cost Estimates for the EDSTAC's Proposed Endocrine Disruptor 3 Screening and Testing Assays 4 5 6 This Appendix contains a detailed summary of a Cost Estimate Survey for Endocrine Disruptor 7 Screening and Testing Batteries conducted in May 1998 by Applied Pharmacology and 8 Toxicology, Inc. ( APT). The purpose of the survey was to project costs for conducting the 9 screening and testing batteries recommended by the EDSTAC. 10 11 Based upon Chapter Five   Screening and Testing of the April 3, 1998 EDSTAC Draft Report, 12 and Appendices J, K, L, and what was P ( and is now Q), APT developed detailed protocols for 13 thirteen screening assays and seven tests according to a standardized format. These protocols 14 were sent to 18 toxicology laboratories competent to conduct the types of assays and tests 15 recommended by the EDSTAC. Fourteen laboratories responded by providing cost estimates for 16 one or more of the assays and tests. The results of this cost estimate survey were summarized in 17 Tables 5.6 and 5.7 in Chapter Five of the final EDSTAC report. A complete copy of the cost 18 estimate survey, including protocols for the thirteen screening assays and seven tests, is included 19 in the EDSTAC docket. 20 21 All materials related to APT's cost estimate survey except the twenty detailed protocols are 22 contained in this Appendix, including: an example of the cover letter that accompanied the 23 survey; the cost estimate form; and the final report. The final report describes the survey design, 24 summarizes the results in Tables 1  3, and provides the individual cost estimates in its own 25 Appendices A and B. 26 27 28 EDSTAC Final Report Chapter Six Appendices August 1998 1 T  Appendix T 1 2 Summary of EPA's September 1997 Outreach Questionnaire 3 4 Following the establishment of the Endocrine Disruptor Screening and Testing Advisory 5 Committee ( EDSTAC), the Communications and Outreach Work Group ( COWG) developed a 6 mailing list and an Outreach Questionnaire which was disseminated to over 1,500 addressees. 7 This Questionnaire was created in an effort to obtain information regarding the public's interest in 8 the EDSTAC and its activities during the Committee's tenure, as well as to assist in future 9 outreach efforts conducted by the U. S. Environmental Protection Agency ( EPA/ the Agency). 10 The information received in response to this questionnaire will assist the Agency in determining 11 the most effective way( s) to communicate with those individuals and organizations interested in 12 the Endocrine Disruptor Screening and Testing Program that EPA will implement following the 13 EDSTAC process. 14 15 EPA received a total of 142 responses to the Questionnaire from the targeted recipients ( a 16 response equal to 9.4%). A break down of respondents by their organizations ( using categories 17 as described in the Questionnaire with additions in some cases) is as follows: 18 21 with Advocacy Organizations 12 with Professional Organizations 2 with Environmental Justice Organizations 18 with Educational Organizations 15 with Government Organizations 6 with For Profit Organizations 10 with Non Profit Organizations 11 with Chemical/ Pesticide Producer Organizations 3 with Chemical/ Pesticide User Organizations 4 with Small Businesses 4 with News Organizations 4 with Law Firms 5 with Trade Associations 3 with Consulting Organizations 2 Medical Doctors 1 Student 1 Zoological Organization ( Zoo) 1 " General Public" Individual 20 with Multiple Designations of: Advocacy/ Environmental Justice/ Educational/ Non Profit Organizations EDSTAC Final Report Chapter Six Appendices August 1998 2 T  Summary of Responses:

1. All respondents were interested in receiving future information regarding the EDSTAC and its activities.

2. Respondents were asked to select their top three choices indicating what types of materials they were interested in receiving regarding EDSTAC activities, and the best vehicles for receiving those materials. [ Note: Some respondents made three choices; some made more than three; and some made less than three choices.]

The types of materials most useful for respondents to receive regarding the EDSTAC were as follows:

· 118 for updates on the progress of the EDSTAC as a whole · 72 for updates on the progress of the EDSTAC work groups · 103 for fact sheets on endocrine disruptors · 42 for resources to seek out additional information on endocrine disruptors · 78 for summary memos from the public EDSTAC meetings · Other: 1 wanted scientific summaries 1 wanted minutes of meetings with copies of documents that were distributed 1 wanted on going and planned wildlife and human studies According to the responses received, the types of vehicles that would be best for respondents to receive information regarding EDSTAC's activities were as follows:


[Federal Register Volume 86, Number 183 (Friday, September 24, 2021)]
[Notices]
[Page 53092]
[FR Doc No: 2021-20712]

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DEPARTMENT OF HOMELAND SECURITY

Federal Emergency Management Agency

[Internal Agency Docket No. FEMA-4535-DR; Docket ID FEMA-2021-0001]

Wyoming; Amendment No. 6 to Notice of a Major Disaster
Declaration

AGENCY: Federal Emergency Management Agency, DHS.

ACTION: Notice.

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SUMMARY: This notice amends the notice of a major disaster declaration
for the State of Wyoming (FEMA-4535-DR), dated April 11, 2020, and
related determinations.

DATES: This amendment was issued August 5, 2021.

FOR FURTHER INFORMATION CONTACT: Dean Webster, Office of Response and
Recovery, Federal Emergency Management Agency, 500 C Street SW,
Washington, DC 20472,<PHONE_NUMBER>.

SUPPLEMENTARY INFORMATION: The notice of a major disaster declaration
dated April 11, 2020 for the State of Wyoming is hereby amended to
include the Hazard Mitigation Grant Program.

    All areas within the State of Wyoming are eligible for
assistance under the Hazard Mitigation Grant Program.

    The following Catalog of Federal Domestic Assistance Numbers
(CFDA) are to be used for reporting and drawing funds: 97.030,
Community Disaster Loans; 97.031, Cora Brown Fund; 97.032, Crisis
Counseling; 97.033, Disaster Legal Services; 97.034, Disaster
Unemployment Assistance (DUA); 97.046, Fire Management Assistance
Grant; 97.048, Disaster Housing Assistance to Individuals and
Households In Presidentially Declared Disaster Areas; 97.049,
Presidentially Declared Disaster Assistance--Disaster Housing
Operations for Individuals and Households; 97.050, Presidentially
Declared Disaster Assistance to Individuals and Households--Other
Needs; 97.036, Disaster Grants--Public Assistance (Presidentially
Declared Disasters); 97.039, Hazard Mitigation Grant.

Deanne Criswell,
Administrator, Federal Emergency Management Agency.
[FR Doc. 2021-20712 Filed 9-23-21; 8:45 am]
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[Federal Register Volume 86, Number 138 (Thursday, July 22, 2021)]
[Notices]
[Pages 38730-38731]
[FR Doc No: 2021-15610]

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DEPARTMENT OF HOMELAND SECURITY

Federal Emergency Management Agency

[Docket ID: FEMA- 2021-0013; OMB No. 1660-0002]

Agency Information Collection Activities: Submission for OMB
Review; Comment Request; Disaster Assistance Registration

AGENCY: Federal Emergency Management Agency, Department of Homeland
Security.

ACTION: 30 Day notice of renewal and request for comments.

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SUMMARY: The Federal Emergency Management Agency (FEMA) will submit the
information collection abstracted below to the Office of Management and
Budget for review and clearance in accordance with the requirements of
the Paperwork Reduction Act of 1995. The submission will describe the
nature of the information collection, the categories of respondents,
the estimated burden (i.e., the time, effort and resources used by
respondents to respond) and cost, and the actual data collection
instruments FEMA will use.

DATES: Comments must be submitted on or before August 23, 2021.

ADDRESSES: Written comments and recommendations for the proposed
information collection should be sent within 30 days of publication of
this notice to www.reginfo.gov/public/do/PRAMain. Find this particular
information collection by selecting ``Currently under 30-day Review--
Open for Public Comments'' or by using the search function.

FOR FURTHER INFORMATION CONTACT: Requests for additional information or
copies of the information collection should be made to Director,
Information Management Division, email address<EMAIL_ADDRESS>or Brian Thompson, Supervisory
Program Specialist, FEMA, Recovery Directorate,<PHONE_NUMBER>.

SUPPLEMENTARY INFORMATION: This proposed information collection
previously published in the Federal Register on April 12, 2021, at 86
FR 19001 with a 60 day public comment period. No comments were
received. The purpose of this notice is to notify the public that FEMA
will submit the information collection abstracted below to the Office
of Management and Budget for review and clearance.

Collection of Information

    Title: Disaster Assistance Registration.
    Type of information collection: Extension, without change, of a
currently approved information collection.
    OMB Number: 1660-0002.
    Form Titles and Numbers: FF-104-FY-21-123 (formerly FEMA Form 009-
0-1T (English)), Tele-Registration, Disaster Assistance Registration;
FF-104-FY-21-1123-A (formerly FEMA Form 009-0-1T (Spanish)), Tele-
Registration, Registro Para Asistencia De Desatre; FF-104-FY-21-123-
COVID-FA (formerly FEMA Form 009-0-1T-COVID-FA (English)), Tele-
Registration, COVID-19 Funeral Assistance; FF-104-FY-21-125 (formerly
FEMA Form 009-0-1Int (English)), Internet, Disaster Assistance
Registration; FF-104-FY-21-125-A (formerly FEMA Form 009-0-2Int
(Spanish)), Internet, Registro Para Asistencia De Desastre; FF-104-FY-
21-122 (formerly FEMA Form 009-0-1 (English)), Paper Application/
Disaster Assistance Registration; FF-104-FY-21-122-A (formerly FEMA
Form 009-0-2 (Spanish)), Solicitud en Papel/Registro Para Asistencia De
Desastre; FF-104-FY-21-128 (formerly FEMA Form 009-0-3 (English)),
Declaration and Release; FF-104-FY-21-128-A (formerly FEMA Form 009-0-4
(Spanish)), Declaraci[oacute]n Y Autorizaci[oacute]n; FF-104-FY-21-127
(formerly FEMA Form 009-0-5 (English)), Manufactured Housing Unit
Revocable License and Receipt for Government Property; FF-104-FY-21-
127-A (formerly FEMA Form 009-0-6 (Spanish)), Las Casas Manufacturadas
Unidad Licencia Revocable y Recibo de la Propiedad del Gobierno;
Request for Information (RFI).
    Abstract: The forms in this collection are used to obtain pertinent
information to provide financial assistance, and if necessary, direct
assistance to eligible individuals and households who, as a direct
result of a disaster or emergency, have uninsured or under-insured,
necessary or serious expenses they are unable to meet. This extension,
without change, will also support the continued ability to provide
COVID-19 Funeral Assistance to individuals who responsible for a
deceased individual's funeral expenses.
    Affected Public: Individuals or Households.
    Estimated Number of Respondents: 2,004,488.
    Estimated Number of Responses: 2,004,488.
    Estimated Total Annual Burden Hours: 622,707.
    Estimated Total Annual Respondent Cost: $24,441,251.
    Estimated Respondents' Operation and Maintenance Costs: $0.
    Estimated Respondents' Capital and Start-Up Costs: $0.
    Estimated Total Annual Cost to the Federal Government:
$203,187,715.

Comments

    Comments may be submitted as indicated in the ADDRESS caption
above. Comments are solicited to (a) evaluate whether the proposed data
collection is necessary for the proper performance of the agency,
including whether the information shall have practical utility; (b)
evaluate the accuracy of the agency's estimate of the burden of the
proposed

collection of information, including the validity of the methodology
and assumptions used; (c) enhance the quality, utility, and clarity of
the information to be collected; and (d) minimize the burden of the
collection of information on those who are to respond, including
through the use of appropriate automated, electronic, mechanical, or
other technological collection techniques or other forms of information
technology, e.g., permitting electronic submission of responses.

Millicent L. Brown,
Senior Manager, Records Management Branch, Office of the Chief
Administrative Officer, Mission Support, Federal Emergency Management
Agency, Department of Homeland Security.
[FR Doc. 2021-15610 Filed 7-21-21; 8:45 am]
BILLING CODE 9111-23-P