NR481364.oi

TITLE

Ortho-Phenylphenol [CAS 90-43-7]: Submission Providing an Evaluation of

Other Scientifically Relevant Information (OSRI) on Ortho-Phenylphenol

(OPP) Considered as Fulfilling Some of the Endpoints Defined in the Tier 1

EDSP Test Battery

DATA REQUIREMENT

EDSP Test Order/DCI Number EDSP-064103-225, EDSP-064103-226

Issued 14, January, 2010

AUTHORS

Pam Spencer, Ph.D., D.A.B.T.

David Geter, Ph.D.

Allen Poole, Ph.D.

Norbert Caspers, Ph.D.

Gisela Stropp, Ph.D.

SPONSOR/SUBMITTER

Lanxess Corporation, Pittsburgh, Pennsylvania, USA

The Dow Chemical Company, Midland, Michigan, USA tcctt c

c c c

e t c

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STATEMENT OF DATA CONFIDENTIALITY CLAIMS

No claim of confidentiality is made for any information contained in this

document on the basis of its falling within the scope of FIFRA, Section

10(d)(l)(A)(B), or (C). This statement supersedes any other confidentiality

claims appearing on title pages of study reports.

Consortium Representative:

Stan Oslosky

LANXESS Corporation

Regulatory Manager

Signature Date: 22 June 2010

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GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT

This report is not subject to the principles of 40 CFR 160, Good Laboratory

Practices Standards (FIFRA), as promulgated in Federal Register, final rule,

August 17, 1989.

Consortium Representative:

Stan Oslosky

LANXESS Corporation

Regulatory Manager

Signature Date: 22 June 2010

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1. Summary

The purpose of this document is to evaluate if any data from the variety of in vitro and in

vivo toxicological assays conducted on OPP are functionally equivalent to some of the

Tier 1 EDSP assays. Current in vitro data from studies using recombinant yeast cells

transfected with human estrogen receptor (ER), human breast cells (E-screen), cells

transfected with the Vit-Luc reporter gene, and ToxCast® do indicate some weak

estrogenic activity with a relative potency several orders of magnitude lower than

estradiol. The low potency is reflected in results from combined chronic and

carcinogenicity assays which have demonstrated a lack of any histopathological changes

in organs and tissues potentially susceptible to hormone active agents (e.g. adrenals,

cervix, epididymis, mammary gland, ovaries, thyroid, parathyroid, prostate, seminal

vesicles, testes and uterus). In addition 2-generation reproductive assays and

developmental assays in rat and rabbit, (all conducted using guideline acceptable doses of

OPP) also failed to find any evidence of specific toxicity related to hormone activity.

Studies in non-mammalian species including fish (vitellogenin measurements) and

invertebrates (reproductive parameters) have found no evidence of adverse hormonal

effects.

Based on the information presented in this document it is proposed that the available data

on OPP described herein are functionally equivalent to those data which will be

generated by conducting all of the Tier 1 EDSP assays except for the amphibian

metamorphosis (frog) assay which will be conducted to aid evaluation of the substance.

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2. Overview of Existing Data on OPP Proposed as Representing "Other

Scientifically Relevant Information" (OSRI) Considered to be

Functionally Equivalent to Selected Endpoints Included in the Tier 1

EDSP Assay Requirements

2.1 In vitro screening studies

2.1.1 Estrogen receptor competitive binding assays

The relative binding affinity of OPP has been tested in an Estrogen receptor (ER)

competitive binding assay using uterine cytosol preparations from adult Sprague-Dawley

rats. Cytosol preparations were incubated with various concentrations of OPP. No ICso

value could be established'by OPP concentrations up to 1x104 M. OPP was classified as a

non-binder in this assay [Blair et al., 2000].

2.1.2 Recoinbinant yeast assays

Recombinant yeast cells transfected with human ER were used to test estrogen-like

response of OPP. Concentrations ranged from 200 nM to 800 uM OPP. The estrogen-like

response of OPP was ca. Ixl06-fold less when compared to the activity of 17(3-estradiol

[Routledge and Sumpter, 1997].

In another assay the activity of OPP was tested in yeast cells expressing a protein

carrying the hormone binding domain of the human ER. Concentrations of 10 nM, 10 uM

or 1 mM OPP did not induce any response in this assay, while diethylstilbestrol was

active at a concentration of 10 nM [Rehmann et al, 1999].

A further assay was conducted in yeast cells transfected with human ERa. Only relative

potencies are presented which were calculated by dividing the concentration of the test

chemical required to produce a half-maximal response by the concentration of 170-

estradiol required to produce the same response. The relative potency of OPP was 2xl06

times less than the potency of 17p-estradiol [Miller et al, 2001].

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2.1.3 Mammalian cell culture assays

The estrogen-like activity of OPP was tested in the "E-Screen" using MCF-7 human

breast cancer cells. In these cells proliferation can be stimulated by addition of estrogens.

The concentration of OPP needed for a proliferative response was 106 times higher than

the concentration of 1 ?p-estradiol needed for proliferative response. The maximum cell

yield by OPP was 30% when compared to the maximum cell yield by 17p-estradiol [Soto

etal., 1997].

In another assay using MVLN cells (MCF-cells stably transfected with the Vit-Luc

reporter gene), the estrogen specific transcription activity of OPP was measured by the

activity of luciferase in cell lysate. The relative estrogenic potency of OPP was 5.4xl03

times less when compared to 17)3-estradiol. The maximum relative luciferase activity was

23% of the luciferase activity achieved by 17p-estradiol [Itoh et al., 2000].

2.1.4 US EPA ToxCast® Program Results of OPP Relevant to

Endocrine Activity.

2.1.4.1 Introduction

ToxCast® is a US Environmental Protection Agency program that includes a battery of

in vitro, high-throughput screening (HTS) assays to analyze chemicals for potential

toxicity. Approximately 69 chemicals have undergone HTS under the ToxCast®

program, the majority of these being pesticides or biocides. The biocide 2-phenylphenol

(OPP) has current requirements for EDSP Tier 1 screening. These assays comprise a

battery of 11 assays that EPA will use to assess the potential interaction of OPP with the

estrogen, androgen, or thyroid (EAT) hormonal systems and target tissues. Within the

ToxCast® database, several assays examine endpoints in the EAT system and could be

utilized as other scientifically relevant information (OSRI) for submittal in lieu of or to

supplement Tier 1 required tests. The ToxCast® data for OPP is publically available at

the EPA ToxCast® website (www.epa.gov/ncct/ToxCast®/index.html). This review was

developed to evaluate the data deemed most relevant to potential endocrine modulation

and the Tier 1 Test Battery and the results are specific to OPP. This review describes

briefly the HTS assay technologies incorporated in ToxCast®, provides an overview of

the HTS assays most relevant to potential EAT endocrine modulation, discusses the Tier

1 screening assays that could be potentially replaced, and provides a specific review of

OPP results based on the ToxCast® data.

2.1.4.2 ToxCast® HTS Test Battery and Relevance to Potential Endocrine Modulation

The ToxCast® HTS test battery includes nine separate in vitro assay technologies:

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1. Cell-free HTS assays to measure binding constants and enzyme inhibition values;

2. Cell-based HTS assays to measure binding constants and enzyme inhibition values;

3. High content cell-imaging assays to test for effects on a range of phenotypes in either

the human hepatoma cell line HepG2 or rat primary hepatocytes;

4. Gene expression in human primary hepatocytes, focused on Phase I and II metabolic

enzymes and transporters;

5. Multiplex transcription reporter assays to evaluate 48 transcription factor binding sites;

6. Biologically multiplexed activity profiling (BioMAP) to characterize effects

potentially relevant to human tissue and inflammatory disease biology;

7. Phase I & II xenobiotic metabolizing enzyme (XME) cytotoxicity assays based on

microarray technology;

8. High-throughput genotoxicity screening based on the GreenScreen high content

GADD45AGFP HTS genotoxicity assay; and

9. Real-time cell electronic sensing (RT-CES) to measure cellular changes in response to

chemicals.

Of these assay technologies, those that provide information most directly relevant to the

issue of potential EAT endocrine modulation and the required Tier 1 in vitro assays

include:

1. The cell-free HTS assays;

2. The cell-based HTS assays;

3. The multiplex transcription reporter assays and;

4. An arcimatase assay.

Each of these assays and their data relevant to potential endocrine modulation is

described below.

Cell-free HTS assays

These assays were run under contract to EPA by Caliper Life Sciences (formally

NovaScreen). Of the 239 endpoints examined, binding to the following receptor proteins

relevant to the endocrine system was evaluated:

1. Bovine estrogen receptor from uterine membrane (bER);

2. Human estrogen receptor from MCF-7 cells (hER);

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3. Bovine progesterone receptor from uterine membrane (bPR);

4. Human progesterone receptor from T47-D cells3 (hPR);

5. Rat androgen receptor (rAR; recombinant protein containing ligand binding domain

only);

6. Human androgen receptor from LnCAP cells (hAR);

7. Rat thyrotropin-releasing hormone receptor from forebrain membranes (rTRH); and

8. Human thyroid hormone receptor-alpha (hTRa; recombinant).

All of these proteins are nuclear receptors, with the exception of the rat thyrotropinreleasing hormone receptor (rTRH), which is a G-protein coupled receptor. Additionally,

all of these, with the exception of the hTRa, were evaluated in competitive binding assays

that measured the displacement of radiolabeled ligand from the receptor proteins. The

hTRa assay, in contrast, examined receptor activation by measuring the luminescent

output from an hTRa-responsive reporter gene. Inhibition of the Aromatase enzyme

(CYP19A1 or CYPC19) activity was also evaluated.

Cell-based HTS assays

The cell-based assays were conducted by the NIH Chemical Genomics Center (NCGC).

Both agonist and antagonist activity at various nuclear receptors, including the human

androgen receptor (hAR), human ERa (hERa), and human thyroid hormone receptor-B

(hTRB), were examined using a reporter gene assay system.

Multiplex Transcription Reporter Assays

These assays were performed by Attagene, Inc. using a multiple reporter transcription

unit (MRTU) library consisting of 48 transcription factor binding sites transfected into

HepG2 cells (Martin et al., 2010). Cis-acting reporter genes examined the activities of

transcription factors endogenous to the HepG2 cells. Trans genes evaluated the activities

of exogenous, chimeric proteins. Each chimera was comprised of the ligand-binding

domain of a nuclear receptor and a yeast Gal4 DNA-binding domain. When activated,

these chimeric proteins activated transcription of a reporter gene expressed downstream

of a 5X-UAS-TATA promoter. In all cases, the reporter gene was identical in sequence

and included a restriction enzyme cleavage site; the position of the restriction site,

however, was unique to each reporter gene.- The system allowed for reporter gene

products to be identified through sequencing and separation via high resolution

electrophoresis.

2.L4.3 Comparison to Tier 1 in vitro Screening Requirements

The ToxCast® data supplement, and in some cases, may fulfill the in vitro data

requirements of the EDSP Tier 1 ER binding assay, AR binding assay, ER transactivation

assay and Aromatase inhibition assay. Additionally, these data provide important OSRI

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that should be considered in a weight-of-evidence evaluation of functionally equivalent

information supporting the absence of need for many of the Tier 1 mammalian in vivo

screening tests.

ER binding assay: The EDSP Tier 1 ER binding assay requires that binding be evaluated

using rat ER derived from uterine tissue taken from ovariectomized Sprague Dawley rats.

In the ToxCast® cell-free HTS assays, binding to bovine and human, but not rat, ER was

evaluated.

Androgen receptor binding assay: The EDSP Tier 1 AR binding assay requires that

binding be evaluated using rat AR derived from prostate tissue taken from castrated

Sprague Dawley rats. In the ToxCast® cell-free HTS assays, binding to both rat and

human androgen receptors were evaluated.

ER transactivation assay: The EDSP Tier 1 ER transactivation assay requires that the

assay be conducted in hER-HeLa-9902 cells, which express the human ERa and have

been stably transfected with a luciferase reporter gene linked to an upstream vitellogenin

estrogen-responsive element. A test chemical is considered positive for ER

transactivation in the Tier 1 assay if the maximum response induced is at least 10% of

that induced by 1 nM 1713-estradiol.

The multiplex transcription reporter assay also examined transactivation of the human

ERa. In this case, the assay is conducted in HepG2 cells (instead of HeLa-9902 cells),

and the output from the reporter gene and how it is measured is different from that

indicated in the EDSP Tier 1 screening assay. If a chemical elicited a low level of

activity at the highest concentration tested in the transactivation or multiplex transcription

reporter assays, it can be assumed that it would be negative according to the EDSP Tier 1

screening criteria.

Aromatase assay: The EDSP Tier 1 Aromatase assay requires that enzyme activity be

evaluated using human recombinant microsomes containing CYP19 plus reductase. In

the ToxCast® cell-free HTS assay, human Aromatase activity was evaluated using

di(benzyloxymethoxy)fluorescein, which produces a fluorescent product as the enzyme

substrate.

It should be noted that the ToxCast® HTS data set includes information that should be

useful to submit as OSRI, and in fact, goes beyond the in vitro testing required in the Tier

1 screening program to provide additional information on the potential to interact with

the EAT systems, including thyroid-related receptors, as well as progesterone receptors.

It does not, however, directly address Tier 1 steroidogenesis in vitro test requirements.

2. /. 4.4 ToxCast® Data specific to OPP

Androgen Receptor (AR):

1. ATG_AR_TRANS - no effect

2. NCGC_AR_Agonist - no effect

3. NVS_NR_rAR - no effect

4. NVS NR hAR- no effect

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5. NCGC_AR_Antagonist - no effect

Inhibition of the Aromatase Enzyme:

1. CLZDJJGT1 Al_48 - no effect

Estrogen Receptor (ER):

1. NVS_NR_hER- no effect

2. NVS_NR_bER- no effect

3. NCGC_ERalpha_Agonist - 18.4 and 29.4-fold increase at 34.3 and 76.6 uM

respectively

4. ATG_ERa_TRANS - 2.8 and 6.8-fold increase at 33 and 100 uM respectively

5. ATG_ERE_CIS- no effect

6. NCGC_ERalpha_Antagonist - no effect

Progesterone Receptor (PR):

1. NVS_NR_bPR - no effect

2. NVS_NR_hPR - no effect

Thyroid Hormone Receptor (TR):

1. NCGC_TRbeta_Agonist- no effect

2. ATG_THRal_TRANS - 1.54-fold increase at 100 uM

3. NVS_NR_hTRa- no effect

4. NCGC_TRbeta_Antagonist- no effect

2.1.4.5 Conclusion ofToxCast® endocrine specific data for OPP

OPP did not elicit a response in the AR, Aromatase, or progesterone specific ToxCast®

assays. However, OPP did show ERa binding and transactivation activity and a very

minimal thyroid hormone receptor transactivation response, all at high test

concentrations.

2.2 Results of Studies in Mammalian Species

2.2.1 Repeated Dose (chronic) toxicity Studies in Mammalian Species

In a combined chronic toxicity/oncogenicity study (OECD 453, GLP) reported by Whale

and Christenson (1996), OPP was administered for 2 years to both sexes of F344 rats (50

animals/sex/dose). Males were given OPP at dietary concentrations of 0, 800, 4000 and

8000 ppm, females received 0, 800, 4000 and 10000 ppm. This resulted in an average

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intake of 0, 39, 200 and 402 mg/kg bw/day in males and 0, 49, 248 and 647 mg/kg

bw/day in females.

Mean body weights were decreased in both sexes at mid-dose (5%) and high-dose (11%),

while food consumption remained unchanged. Mortality was increased in high-dose

males. Clinical observations included abnormal urine color and urine stains in mid- and

high-dose groups. Ophthalmology, hematology, and clinical chemistry were not affected

by treatment. Urinalyses showed an increased incidence of blood in the urine of highdose males. No evidence of an alteration in organ weights, attributable to exposure to

OPP, was suggested in either sex at any dose tested. The major toxicological finding was

an increase incidences of urinary bladder neoplasia (papilloma: 12%, transitional cell

carcinoma: 68%) seen in high dose group male rats. In contrast, no neoplastic changes

were observed in females at any dose level. Non-neoplastic lesions occurred in the kidney

of high dose males and females. These effects are not endocrine-related. In examining

this chronic study, no adverse effects on estrogenic, androgenic or thyroid hormone

pathways or histopathological findings were noted in organs and tissues potentially

susceptible to endocrine active compounds (e.g. adrenals, cervix, epididymis, mammary

gland, ovaries, parathyroid, pituitary, prostate, seminal vesicles, testes, thyroid and

uterus).

In another combined chronic toxicity/oncogenicity study (OECD 453, GLP), reported by

Quast and McGuirk, (1995) B6C3Fi mice (50/sex/group) were fed diets supplying 0, 250,

500 or 1000 mg OPP/kg bw/day for 2 years. Clinical signs and mortality were unaffected

by treatment. Body weights and body weight gains of all treated groups were decreased

(6-20 % and 10-38 %, respectively), except in 250 mg/kg males. Hematology, clinical

chemistry, and urinalyses were unaffected by treatment. Treatment was associated with

liver enlargement and an increase in the incidence of hepatocellular adenoma occurring in

male mice treated at 500 and 1000 mg/kg. Female mice showed no increases in incidence

of hepatic tumors at any dose level. Both sexes showed histopathological changes in the

liver suggestive of an adaptive change to the increased metabolic demand required to

metabolize and clear OPP from the body.

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In a similar manner to the rat chronic study, mice treated with high doses of OPP showed

no evidence of toxicological effects (including weight changes or histopathology) in

organs and tissues potentially susceptible to endocrine active compounds (e.g. adrenals,

cervix, epididymides, mammary glands, ovaries, oviducts, parathyroid, pituitary, prostate,

seminal vesicles, testes, thyroid, uterus, vagina). Furthermore, in a 2 year dermal study in

mice, OPP also failed to provide any evidence of endocrine toxicity (NTP Technical

Report, 1986).

A 1 year study in Beagle dogs (COSSE et al., 1990) given 0, 30, 100, or 300 mg OPP/kg

bw/day in a peanut oil solution via gastric intubation 5 days per week for 1 year (EPA

Guideline 83-1, GLP) again failed to show any evidence of adverse effects on tissues and

organs which would be expected to be sensitive to the action of a hormone active

substance (e.g. adrenals, cervix, epididymides, mammary glands, ovaries, oviducts,

parathyroid, pituitary, prostate, seminal vesicles, testes, thyroid, uterus, vagina).

In summary, chronic studies in the rat, mouse and Beagle dog have repeatedly shown no

toxicological effects associated with modulating the estrogen, androgen or thyroid

hormone pathways.

2.2.2 Reproductive toxicity studies (Fertility)

OPP was investigated in a 2-generation reproduction study (EPA Guideline 83-4; GLP)

with Sprague-Dawley rats (Eigenberg, 1990). In this investigation OPP was administered

in the diet at doses of 35, 125, and 457 mg/kg bw/day based on the mean analytical

concentrations in the diet. Treatment started 15 weeks prior to mating and continued

throughout mating, gestation and lactation, with further exposure of the offspring after

weaning. Treatment produced an increased incidence of calculi in the kidney and/or

urinary bladder in male rats in the 140 and 490 mg/kg groups. Transitional cell

hyperplasia/p.apillomatosis in the urinary bladder was significantly elevated in the 490

mg/kg FO males and females and in the 490 mg/kg Fj males. There were no treatmentrelated effects on reproduction or other parameters which might be considered

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susceptible to effects caused by exposure to a hormone active agent (e.g.estrous cyclicity,

reproductive parameters).

In a second 2-generation study with two matings per generation (Eigenberg, 1995), OPP

was administered in the diet to Sprague-Dawley rats (30/sex/group) at dose levels of 0,

20, 100 and 500 mg/kg bw/day (EPA Guideline 83-4, GLP). The application started 10

weeks prior to mating and continued throughout mating, gestation and lactation, with

further exposure of the offspring after weaning. P adults were mated to produce Fia and

Fib litters and FI adults (randomly selected Fib pups) were mated to produce Fia and F2b

litters.

Similar effect to the previous reproductive study were seen such as urinary calculi with

transitional cell hyperplasia (simple and/or nodular/papillary), calculi, and chronic

inflammation in the urinary bladder as well as dilatation and hyperplasia of the ureter in

males of the 500 mg/kg group. There were no test compound-related effects on

reproductive parameters. No effects were seen on litter size, gender, the number of

stillborn pups, pup viability, clinical signs of pups, nor gross pathology of the pups.

Estrous cyclicity was not affected in both FQ and FI. As in the previous 2-generation

reproduction study, this study also showed no treatment-related effects on reproduction or

other parameters associated with altered endocrine activity. Together, these 2-generation

studies showed no evidence for a substance- or dose-related endocrine effect of OPP.

2,2.3 Developmental toxicity studies

The developmental toxicity of OPP was tested on pregnant Wistar rats (11-20/group)

using doses of 0, 150, 300, 600 and 1200 mg/kg bw/day (Kaneda et al, 1978). The test

substance was administered daily by gavage on days 6 to 15 of gestation. The females

were sacrificed on day 20 of gestation for determination of the number of implantations,

as well as the number of dead and living fetuses. Living young were inspected externally

for anomalies and weighed individually. Afterwards fetuses were examined for skeletal

or internal anomalies.

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While treatment at 300 mg/kg and above was associated with ataxia and retardation of

body weight gain, no differences from the controls were recorded in relation to the

number of implantations, the number of living fetuses, resorption rates or fetus weights.

At 600 mg/kg and above, the resorption rate was increased while the fetal body weight

was decreased. These maternal and fetal toxic effects were not considered indicative of

any specific endocrine toxicity.

In another developmental toxicity study OPP was administered to pregnant SpragueDawley rats (John et al., 1981). The rats (35 in the control group, 25-27 in the treatment

groups) were given the test substance by gavage at dosages of 0, 100, 300 and 700 mg/kg

bw/day on days 6 to 15 of gestation. The animals were sacrificed on gestation day 21.

Body weight gain and liver weight of dams were measured. Furthermore, implantation,

live fetuses, resorptions, fetal sex ratio, fetal body weight and fetal crown-rump length

were determined. All fetuses were examined externally and skeletally, and the soft tissue

of approximately one-third of the fetuses was examined. Body weight gain of the dams

receiving a dose of 700 mg/kg/day was significantly reduced on the first 3 days of

administration (day 6 to 9 of gestation), as was the food intake on days 9 thru 11. The

transitory reduction in body weight gain and the reduced food intake after 700 mg/kg

were regarded as treatment-related. Absolute liver weight but not relative (to body

weight) liver weight was decreased at 700 mg/kg. There was no treatment-related effect

on the number of implantations, the number of pups per litter, the frequency of resorbed

embryos or the body weight of the fetuses. As in the first developmental assay there was

no evidence of a specific endocrine activity.

A developmental study conducted in groups of 16-24 pregnant New Zealand White

rabbits in which OPP was administered via gavage on days 7-19 at dose levels of 25, 100

or 250 mg/kg bw/day (OECD 414, GLP) also failed to find any evidence of .

developmental, teratogenic or hormonal effects (Zablotny etal.,1991]

In summary results of reproduction and developmental studies with OPP have failed to

find any toxicological effects which might be associated with modulating effects on

estrogen, androgen or thyroid hormone pathways.

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2.3 Studies in Fish and Invertebrates

2.3.1 Fish Study

A study investigating the effects of OPP on reproduction in fathead minnows

(Pimephales promelas) has been carried out (AstraZeneca 2002) using the pair breeding

assay procedure (Harries et al 2000). The study assessed the effects of 21 day exposure to

Preventol O extra (99.9% purity OPP) via a flow through system at nominal

concentrations of 1.0, 5.0, 50 and 500 ug L"1 (actual concentrations = not detectable, 4.0,

36 and 293 jag L"). A positive control of 10 ng L" 17a-ethinylestradiol was also used.

The test was initiated with mature adults that had a record of reproductive success as

measured by fecundity (number of spawnings, number of eggs, number of eggs per

brood) and by embryo viability (hatchability of eggs). This was established during a preexposure period of 21 days in the same system/tanks utilised for the subsequent chemical

exposure. Each replicate tank contained a pair of male and female fathead minnows and

eight breeding pairs were used at each concentration. Measurements of fecundity were

assessed daily and the viability of resultant embryos was assessed in animals held in the

same treatment regime to which the adults were exposed. At the conclusion of the test,

blood samples were collected from the adults for determination of vitellogenin and the

gonads were sampled for measurement of the gonadosomatic index (GSI) and

histological analyses (if required).

The endpoints assessed were number of spawnings, egg production, egg batch size, GSI

(males), GSI (females) Vitellogenin (males), and Vitellogenin (females), all in FO fish,

and egg hatch in Fl fish. OPP did not exhibit any adverse effects on these reproductive

parameters up to a measured test concentration of 36 |o,g L"1 (nominal test concentration

of 50 ng L"1). With regard to the induction of vitellogenin and changes in gonadosomatic

index and early indication of possible oestrogenic effects no substance-related effects

were noted even at the highest concentration (293 u£ L"1) compared to the effects

observed in the positive control 17a-ethinylestradiol. From the data the overall no

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observed effect concentration (NOEC) has been reported as 36 ug L"1

 based on measured

concentrations (50 fag L"1 based on nominal concentrations). The observation that no

effects on vitellogenin induction and gonadosomatic index occurred at 293 fig L"1

(highest exposure level) underpins that the overall effects in this tests were obviously not

oestrogen-mediated.

2.3.2 In vertebrate Studies

Two 21 day reproduction tests using Daphnia magna have been carried out which

investigated the effects of OPP on the survival of adult Daphnia and juvenile production

of surviving Daphnia adults (Bayer 1989, 2001). In the original study the NOEC for

effects on juvenile production was 0.075 mg L"1 (based on nominal concentrations. In the

later juvenile production study no mortality of parental females was observed even at the

highest test concentration (0.1 mg L"1) and the decrease in juvenile production at 0.1 mg

L"1 was only 18.5% (118 juveniles per female compared to 140 juveniles per female in

the control). The NOEC and LOEC values reported in the study were 0.01 and 0.03 mg

L"1 respectively based on nominal concentrations (0.009 and 0.022 mg L"1 based on

measured concentrations).

From these tests, no information is available on the mechanism of action for the observed

toxicity effects.

3 Toxicological Data Assessment & Proposal for Further Work

Given the extensive toxicological data base on OPP it is pertinent to determine what

information contained within the data set can be considered as functionally equivalent to

the information which will be developed through conducting certain investigations

described in the Tier 1 endocrine screening assays. The availability of "Other

Scientifically Relevant Information" (ORSI) within the OPP data set-can provide an

effective substitution for selected Tier 1 screening assays thus providing a mechanism to

avoid unnecessary animal testing.

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3.1 Evaluation of Need for In Vitro Assays,

namely Estrogen Receptor Binding Assay Using Rat Uterine Cytosol, Estrogen

Receptor Transcriptional Activation (Human Cell Line, Androgen Receptor

Binding (Rat Prostate Cytosol) and Steroidogenesis (Human Cell Line-H295R)

For OPP, in vitro screening studies have shown no or only weak estrogen-like activity in

estrogen-receptor binding assays, recombinant yeast assays, mammalian cell culture

assays, and the ToxCast® assays. In those experiments where weak activity of OPP was

found, the potency of OPP was ca. 106-fold less than the potency of the endogenous

estrogen 17p-estradiol and very minimal thyroid receptor transactivation, whereas there

was no effect in assays related to androgen receptor, progesterone receptor, aromatase as

well as thyroid.

With the availability of the current in vitro data and the results from chronic dose studies

and reproductive and developmental assays that covers reproductive organs as well as

organs related to the thyroid system it is proposed that conducting the estrogen and

androgen receptor binding, the estrogen receptor transcriptional activation, the

Steroidogenesis, and Aromatase assays will provide little or no additional useful

information about the potential hormonal properties and subsequent potential health and

environmental risks posed by exposure to OPP.

3.2 Evaluation of Need for Uterotrophic or Female Pubertal assays

Based on results from chronic bioassays, 2-generation reproductive assays and

developmental assays there are no findings or indications that OPP is producing

toxicological effects by disrupting or interfering with the estrogenic hormone system in

females. For example the results from two 2-generation reproduction studies with OPP

show no evidence of adverse effects associated with estrogenicity with end points such

as estrous cyclicity, ovarian follicle count, mating index, corpora lutea, anogenital

distance, epididymis sperm count, fertility, mating index and vaginal being unaffected

by treatment. In addition no exposure related increases in mammary or ovarian tumors

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or indeed any histopathological effects of any reproductive or hormone sensitive tissues

have been seen in oncogenicity studies conducted in both the rat and the mouse. There

has been no evidence of thyroid effects associated with exposures to OPP with treated

animals showing no changes in either thyroid histopathology or weight following life

time exposures to doses producing systemic toxicity. It should be noted the potential for

OPP to cause thyroid effects will be further evaluated in the amphibian metamorphosis

assay which will be conducted.

Overall the available data from chronic, reproduction and developmental studies provide

no evidence or indications that exposures to OPP has any effect in females on estrogen

or indeed androgen sensitive parameters. This conclusion is in line with the scientific

evaluation of OPP within an evaluation of 12 substances in the context of endocrine

disrupter priority list of action by the EU Commission (2002). Based on this evaluation

the data from chronic, reproductive and developmental assays are proposed as OSRI for

the uterotrophic and female pubertal assays.

3.3 Evaluation of Need for Hershberger or Male Pubertal assays

The results of chronic toxicological investigations and reproductive and developmental

assays have shown no pattern of effects which might be interpreted as indicating OPP

has androgenic or anti-androgenic properties. The results of the 2 year carcinogenicity

study showed no evidence of treatment related histopathological changes in testis,

epididymis, prostate, seminal vesicles, ovary or uterus. For example there was no

evidence of carcinoma of the testes or Leydig cell hypertrophy/hyperplasia with no

reports of hypertrophy of the ovary or cystic endometrial hyperplasia of the uterus. All

adverse effects elicited by exposures to androgens and anti-androgenic substances.

Similarly results from the 2-generation reproductive studies provide no evidence for

effects on preputial separation, sperm counts, vaginal patency etc parameters which

might be expected to be affected by androgens. This conclusion is in line with the

scientific evaluation of OPP within an evaluation of 12 substances in the context of

endocrine disrupter priority list of action by the EU Commission (2002).

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There, has been no evidence of thyroid effects associated with exposures to OPP with

treated animals showing no changes in either thyroid histopathology or weight following

life time exposures to doses producing systemic toxicity. It should be noted the potential

for OPP to cause thyroid effects will be further evaluated in the amphibian

metamorphosis assay which will be conducted.

Overall it is proposed the existing 'data on OPP provide the functional equivalence to the

Hershberger and male pubertal assays. Based on this evaluation the data from chronic,

reproductive and developmental assays are proposed as OSRI for the Hershberger and

male pubertal assays.

3,4 Evaluation of Need for Fish Assay

The available fish reproduction screening study which was carried out at the AstraZeneca

laboratories with fathead minnows (2002) was intentionally designed to cover a broad

range of 8 ecotoxicological endpoints, considered to be vital for the reproductive success

of aquatic vertebrates, and. adequately reflecting potential risks caused by OPP to

reproductive features of fish . The results indicate that no adverse effects on reproductive

health and hatchbility of fish are expected to occur at OPP concentrations equal to, or

lower than 36 ug L'1 (based on measured concentrations).

As no effects on vitellogenin induction and gonadosomatic index became evident in this

study, even at the highest exposure concentration (0.29 mg L"1), it can be derived that the

effects of OPP on reproductive parameters may not be oestrogen mediated.

This evaluation is shared by the EU Commission in their "Study on the scientific

evaluation of 12 substances in the context of endocrine disrupter priority list of actions"

(2002).

Overall, the available information from the AstraZeneca study (2002), and the evaluation

of the study results by the EU Commission (2002) indicate that OPP does not cause any

specific endocrine-mediated adverse effects at concentrations below the threshold

concentration for conventional ecotoxicological endpoints. The AstraZeneca study (2002)

is, in large part, equivalent to the fish short term reproduction guideline study (OPPTS

890.1350) developed for Tier 1 of the US EPA's Endocrine Disrupter Screening

Program. The AstraZeneca study (2002) was conducted prior to the development of the

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OPPTS 890.1350 guideline, but does share the basic study design of the OPPTS

guideline. Minor differences related to vessel size, flow rate, light intensity values etc. are

not considered relevant in the sense of triggering diverging test results. Based on this

evaluation the available fish reproduction study is considered functionally equivalent and

proposed as OSRI for the required Fish Short-term Reproduction Test.

3.5 Summary Overview on Toxicological Data Assessment & Proposal for Further

Work

The following table (Table 1) summarizes the assays described in the EDSP Tier 1 which

1) can be addressed by the toxicological data available currently on OPP, (i.e. the

available data are proposed as being functionally equivalent to the data which will be

produced by conducting the Tier 1 assay), and

2) which do not have functional equivalent endpoints available currently and for which

studies will be performed.

Table 1: Tier 1 Test Battery and Requested EPA Action Based on OSRI Weight of the Evidence.

Endocrine Disrupter Screening Program

Tier 1 Test Guideline

OPPTS 890.1250: Estrogen Receptor Binding

Assay Using Rat Uterine Cytosol (ER-RUC)

OPPTS 890.1300: Estrogen Receptor

Transcriptional Activation (Human Cell Line

(HeLa-9903))

OPPTS 890.1 150: Androgen Receptor Binding (Rat

Prostate Cytosol)

OPPTS 890.1200: Aromatase (Human

Recombinant)

OPPTS 890.1550: Steroidogenesis (Human Cell

Line-H295R)

OPPTS 890. 1 600: Uterotrophic Assay

OPPTS 890.1450: Pubertal Development and

Thyroid Function in Intact Juvenile/ Peripubertal

Requested Action

WAIVER requested based on Functional

Equivalence of OSRI

WAIVER requested based on Functional

Equivalence of OSRI

WAIVER requested based on Functional

Equivalence of OSRI

WAIVER requested based on Functional

Equivalence of OSRI

WAIVER requested based on Functional

Equivalence of OSRI

WAIVER requested based on Functional

Equivalence of OSRI

WAIVER requested based on Functional

Equivalence of OSRI

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Female Rats

OPPTS 890.1400: Hershberger Bioassay

OPPTS 890.1500: Pubertal Development

and Thyroid Function in Intact Juvenile/

Peripubertal Male Rats

OPPTS 890.1100: Amphibian Metamorphosis

(Frog)

OPPTS 890.1350: Fish Short-Term

Reproduction Assay

WAIVER requested based on Functional

Equivalence of OSRI

WAIVER requested based on Functional

Equivalence of OSRI

The OPP test consortium will conduct (no waiver

requested)

WAIVER requested based on Functional

Equivalence of OSRI

Finalization Date: June 16, 2010

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