A predictive data-driven framework for endocrine prioritization: a triazole fungicide case study

Figures & data

Figure 1. Number of HTS assays available for the three triazoles within each endocrine activity hypothesis. The assay endpoints available by estrogen (E), anti-estrogen (AE), androgen (A), anti-androgen (AA), steroidogenesis (S), thyroid receptor (TR), anti-thyroid receptor (ATR) and hepatic catabolism related to thyroid hormone (Liver). Not all triazoles were tested in all assays, leading to minor discrepancies between triazoles for the number of assay endpoints available.

Figure 2. Triadimefon: Parallel comparison of predicted bioactivity and exposure and US EPA chronic exposure estimates and in vivo bioactivity. Annotated to show Bins 1–2. See text, Table 6, and Supplemental File 2 for additional details on AC₅₀ by assay and bin. Dotted line = cytotoxicity caution flag (1.68 μM or 5.13 mkd). LPEAD, lowest potentially endocrine active dose (= 1800 ppm or 100 mkd for triadimefon).

Figure 3. Propiconazole: Parallel comparison of predicted bioactivity and exposure and EPA chronic exposure estimates and in vivo bioactivity. Annotated to show Bins 1–8. See text, Table 7, and Supplemental File 2 for additional details on AC₅₀ by assay and bin. Dotted line = cytotoxicity caution flag (4.6 μM or 4.15 mkd). LPEAD, lowest potentially endocrine active dose (= 2500 ppm or 200.4 mkd for propiconazole).

Figure 4. Myclobutanil: parallel comparison of predicted bioactivity and exposure and EPA chronic exposure estimates and in vivo bioactivity. Annotated to show Bins 1–4. See text, Table 8, and Supplemental File 2 for additional details on AC₅₀ by assay and bin. Dotted line = cytotoxicity caution flag (4.69 μM or 5.13 mkd). LPEAD, lowest potentially endocrine active dose (= 200 ppm or 9.84–12.86 mkd for myclobutanil). For graphing purposes, the midpoint of the interval between 9.84 and 12.86, or 11.35 mkd, was used as the LPEAD.

Table 1. Steady state systemic concentration values used to calculate oral equivalent doses.

PAI	Css95%* (μM)
Myclobutanil	0.570242
Propiconazole	1.108062
Triadimefon	0.327346

* Css95% values (human) equivalent to a 1 mkd dose level, from (US EPA, 2014b, Appendix 3).

Table 2. Predicted human oral exposure range for three triazoles.

PAI	Total populationmedian (mg/kg/d)	Total populationupper 95% (mg/kg/d)
Myclobutanil	9.916 E-8	6.022 E-6
Propiconazole	2.476 E-8	1.766 E-6
Triadimefon	1.7 E-8	1.765 E-6

These exposure predictions reflect the total population. From Wambaugh, et al. (2014).

Table 3. Chronic aggregate exposure estimates from EPA risk assessments.

PAI	Range (min–max) acrosssub-populations (mg/kg bw/d)	Total US general population(mg/kg-bw/d)	Model note	Reference
Myclobutanil	0.002882–0.007569	0.005013	DEEM-FCID ver 2.03 (with refined inputs such as USDA PDP data for three crops and average % crop treated for 25 crops with remainder at 100% crop treated)	USEPA (2007c, 2008)
Propiconazole	0.005620–0.023722	0.007434	None	USEPA (2014d)
Triadimefon	0.000503–0.001108	0.000607	Water model is “Entire Golf Course” (DEEM-FCID). As use is largely restricted to turf-use, chronic exposure is not anticipated	USEPA (2009)

Table 4. EDSP Tier 1 assays plus published OSRI for the three triazoles.

OCSPP guideline	Test name	Method	Myclobutanil	Propiconazole	Triadimefon
890.1100	Amphibian metamorphosis (frog)	21 d continuous flow-through exposure of Xenopus laevis tadpoles and measurement of development and markers of thyroid function	Y	Y	N; Goetz et al. (2007), Wolf et al. (2006), and Rockett et al. (2006)
890.1150	Androgen receptor binding (rat prostate)	20 h exposure using rat ventral prostate as a source of cytosolic AR, measuring displacement of R1881	Y	N; Bauer et al. (2002)supported by Kojima et al. (2004)*	Y
890.1200	Aromatase (human recombinant)	15 min exposure using recombinant human aromatase to measure inhibition of enzymatic conversion of ^3H-androstenedione	Y	N; Ohno et al. (2004),supported by 4 other studies^†	N; USEPA (2007b), Ohno et al. (2004), and Goetz et al. (2009c)
890.1250	Estrogen (receptor Binding (ER-RUC)	16–20 h exposure using rat uterine cytosolic ERα, measuring displacement of 17β-estradiol	Y	Y	Y
890.1300	Estrogen Receptor Transcriptional Activation (HeLa-9903)	20–24 h exposure with measurement of hERα activation as a transcription factor to upregulate luciferase production in stably transfected HeLa cells	Y	N; Kjaerstad et al. (2007); Kojima et al. (2004); Hurst and Sheehan (2003)^‡	Y
890.1350	Fish short-term reproduction (FSTRA)	21 d continuous flow-through exposure of sexually-mature fathead minnows followed by measurement of reproductive success	Y	Y	Y
890.1400	Hershberger (rat)	Oral exposure to castrated male CD rats from PND 55-64, followed by measurement of accessory sex tissue weights	Y	N; Goetz et al. (2007), Kjaerstad et al. (2007) supported by Taxvig et al. (2008) and Tully et al. (2006)	Y
890.1450	Female pubertal (rat)	Oral exposure to juvenile female CD rats from PND 22-42 followed by measurement of clinical chemistry, age and BW at VO, tissue weights (pituitary, thyroid, liver, kidneys, adrenals, uterus, ovaries), histology (uterus, ovary and thyroid, kidney), serum T4 and TSH, and estrous cycle	Y	N; Rockett et al. (2006), supported by: Goetz et al. (2007) and Wolf et al. (2006)	N; Rockett et al. (2006)
890.1500	Male pubertal (rat)	Oral exposure to juvenile male CD rats from PND 23-53, followed by measurement of clinical chemistry, age and BW at PPS, tissue weights (pituitary, thyroid, liver, kidneys, adrenals, testes, and accessory sex organs), and histology (epididymis, testis, thyroid, kidney), and serum T4, TSH and testosterone	Y	N; Goetz et al. (2007),supported by: Tully et al. (2006).	N; Goetz et al. (2007); supported by Goetz et al. (2009c), Tully et al. (2006), and Wolf et al. (2006)
890.1550	Steroidogenesis (human – H295R)	48 hr exposure in the H295R human adrenocortical carcinoma cell line followed by measurement of testosterone and estradiol production in the cell culture medium	Y	N; Goetz et al. (2009) and Kjaerstad et al. (2007)	N; Goetz et al. (2009)
890.1600	Uterotrophic (rat)	Oral exposure to immature female CD rats from PND18-21, followed by measurement of uterine weight.	Y	Y	N; Rockett et al. (2006)
Goetz et al. (2007)	Male reproductive developmental landmarks	Dietary exposure from GD6 to PND120. One ♂ per litter was evaluated at PND1, 22, 50 and 92. Measurements included:• Litter size, gender ratio and wt• AGD at PND0• BW and abs and rel organ wts (liver, testis,ventral prostate, epididymis and seminal vesicle) • Liver histopath• Serum hormone luteinizing hormone• Serum estradiol, testosterone, T4, T3 and TSH• Age and BW at PPS• Sperm count, morphology and motility• Fertility and fecundity	Y	Y; supported waivers for the Hershberger and Female and male pubertal assays	Y; supported waivers for the female and male pubertal assays
Goetz et al. (2009)	Evaluation of steroidogenesis	• H295R steroidogenesis assay with measurement of estradiol, progesterone, and testosterone in the cell culture medium• Adult and neonatal rat in vitro testis culture with measurement of testosterone, androstenedione, 17α-hydroxyprogesterone and progesterone production• Dietary 1800 ppm triadimefon exposure from PND60 to PND90 in adult ♂ rats with measurement of: BW; liver, epididymis, ventral prostate, seminal vesicle, testes, pituitary wts; intratesticular testosterone production; serum testosterone	Y; H295R and testis culture only	Y; H295R only	Y; all three study parts; supported waiver of steroidogenesis and male pubertal assays
Hester and Nesnow (2008)	Gene expression analysis in rat thyroid	30 or 90 d dietary exposure in ♂ rats followed by gene array analysis of thyroid tissue	Y	N	Y; supported waiver for amphibian metamorphosis assay
Kjaerstad et al. (2007)	Anti-androgenic Hershberger Assay	Similar to Rat Hershberger Assay (OCSPP 890.1400)	N	Y; supported waivers for ER-TA, Steroidogenesis, and Hershberger Assays	N
Rockett et al. (2006)	Female reproductive developmental landmarks	Dietary exposure from GD6 to PND92. Measurements included: • Litter size, gender ratio and wt• AGD at PND0;• BW and abs and rel organ wts, inc liver, pituitary, hippocampus, hypothalamus, thyroid, ovaries, drained uterus• Liver, ovary, and thyroid histopath• Serum estradiol levels• Age at VO• Estrous cyclicity	Y	Y; supported waiver for the female pubertal assay	Y; supported waivers for the amphibian metamorphosis, female pubertal, and uterotrophic assays
Taxvig et al. (2008)	Short-term in vivo endocrine studies	• Study 1: rat Hershberger Assay similar to OCSPP 890.1400• Study 2: gestational study: pregnant ♀ dosed by gavage GD7-GD20, with Cesarian section on GD21, followed by analysis of post-implantation loss, AGD, and testicular testosterone in foetuses	N	Y; supported waiver for Hershberger Assay	N
Tully et al. (2006)	Testis and hepatic biomarkers	Oral gavage exposure to adult ♂ rats for 14 d followed by measurements including:• BW & rel organ wts, including liver, spleen, adrenal, testis, epididymis, seminal vesicle, ventral prostate and brain• Liver histopath• Serum testosterone, FSH, LH and estradiol• Sperm morphology and motility• Hepatic and testis gene expression analysis	Y	Y; supported waivers for the Hershberger and male pubertal assays	Y; supported waiver for male pubertal assay
USEPA (2007b)	Aromatase inhibition assay	15 min exposure using recombinant human aromatase to measure inhib of enzymatic conversion of ^3H-androstenedione. This was the US EPA validation study for this assay	N	N	Y; supported waiver of aromatase inhibition assay
Wolf et al. (2006)	Markers of thyroid disruption	Dietary exposure to adult ♂ rats for 4, 30 or 90 d followed by measurements including:• BW and liver wt• Liver and thyroid histopath• Serum T3, T4, TSH and cholesterol• EROD, PROD, MROD and UDPGT activity in liver microsomes	Y	Y; supported waiver for the female pubertal assay	Y; supported waivers for the amphibian metamorphosis and male pubertal assays

The EDSP Tier 1 battery and relevant publications used as OSRI are described. For each of the three triazoles, the EDSP Tier 1 assay data submitted and published OSRI used to fulfill the EDSP data requirements, are described. (Y = yes, study conducted; N = no, study was not conducted; OSRI used in place of the study are cited).

* These studies are described in more detail in Tables 9–11, and represent similar in vitro study designs to assess androgen receptor binding or transactivation.

† These studies are described in greater detail in Tables 9–11, and all represent similar in vitro study designs to evaluate aromatase inhibition.

‡ Kojima et al. (2004) and Hurst and Sheehan (2003) are described in greater detail in Tables 9–11, and represent similar in vitro study designs to evaluate estrogen receptor transactivation.

Table 5. Guideline toxicity studies.

Study type	EPA	OECD	Endpoints of endocrine relevance	Additional considerations
Subchronic rodent	870.3100	408
Subchronic dog	870.3150	409	Following repeat dose exposure at 90 d, measurement of weights (liver, kidneys, adrenals, gonads, brain (rat), thyroid (dog), gross pathology and histopathology of numerous tissues, including endocrine-relevant tissues (pituitary, brain, thyroid/parathyroid, adrenals, testis, ovary, uterus, epididymis, prostate, seminal vesicles, and, if indicated by signs of toxicity, mammary gland), in male and female animals	Weights and pathology, while providing valuable information, do not provide mechanistic information on endocrine system function. Older subchronic rodents studies may or may not have weighed or examined all accessory sex tissues separately
Developmental neurotoxicity (DNT), rat	870.6300	426	Following perinatal exposure (GD6-PND21), measurement of:• Sexual maturity (VO and PPS)• Day of eye opening• Behavioural ontogeny, neurobehavioral assessment, motor activity and coordination, acoustic startle, learning and memory• Brain wt, histopath and morphometry• Serum thyroid hormones (optional)	The DNT exposure examines effects resultant to perinatal exposure only, as dosing does not continue through sexual maturation. Acoustic startle is a relatively insensitive technique to assess hearing changes in rats due to moderate perturbations in thyroid homeostasis. Neurobehavioral endpoints and learning and memory are more sensitive, but may miss minor changes in thyroid function. Similarly, brain morphometry changes may only be sensitive to severe thyroid hormone deprivation; myelin staining, which may aid in detection of thyroid-related neurological changes, is optional. Serum thyroid hormone measurement is not required and not always available
Combined chronic andoncogenicity study, rat	870.4100	452	Following prolonged exposure (1–2 years), measurement of:• Weights (liver, kidneys, adrenals, gonads, brain)• Gross pathology and histopathology of numerous tissues, including endocrine-relevant tissues (pituitary, brain, thyroid/parathyroid, adrenals, testis, ovary, uterus, epididymis, prostate, seminal vesicles, and female mammary gland) in male and female animals	Weights, gross pathology, and histopathology, while providing valuable information, do not provide mechanistic information on endocrine system function. Older studies may or may not have weighed or examined all accessory sex tissues separately
	870.4200	451
	870.4300	453
Oncogenicity study, mouse	870.4200	451
Chronic dog (1 or 2 year exposure)	870.4100	452
Developmental toxicity, rodent/rabbit	870.3700 (83–3 before 1998)	414	Following exposure of dams from implantation to the day prior to expected parturition, measurement of:• Maintenance of pregnancy• Uterus wt• # of implants and corpora lutea• Viability, litter size, sex ratio, fetal BW• Fetal examinations (external, visceral, and skeletal)	Though some aspects of endocrine and reproductive function are assessed by this study design, endocrine assessment of the offspring (F1 generation) is limited
	870.3550	421
	870.3650	422
Multigenerational study, rodent	870.3800 (83–4 before 1998)	416	Exposure of parental (P) generation before and during mating, during pregnancy, and through the weaning of the F1 generation. F1 generation is then exposed during growth, mating, pregnancy, generation of the F2 generation, through F2 weaning• Evaluation of reproductive performance (mating, fertility, time to mating, gestation length, difficulty of parturition)• Litter parameters (offspring viability, litter size, pup body weights and sex ratio) • Histopathology of endocrine-relevant tissues (vagina, uterus, ovaries, testes, epididymides, seminal vesicles, prostate, pituitary and target organs)	Studies run prior to 1998 are unlikely to report all reproductive and accessory sex tissue organ wts, estrous cyclicity, ovarian follicle counts, sperm parameters, puberty onset, thyroid wt/histopath, adrenal wt/histopathology and/or AGD (optionally triggered in F2 offspring)

Table 6. Triadimefon: Endocrine-related HTS bioactivity data.

Triadimefon Screening Information Estimated cytotoxicity limit =1.68 μM
Endocrine activity hypothesis (positives/total # assays)	Assay Endpoint ID	AC50 (μM)	AUC model score
Steroidogenesis (2/2)	NVS_ADME_hCYP19A1	2.07	NA‡
	Tox21_Aromatase_Inhibition	36.84
Estrogenic (4/15)	ACEA_T47D_80hr_Positive	7.94	0.0095 (negative)
	ATG_ERa_TRANS_up	16.46
	ATG_ERE_CIS_up	18.08
	OT_ER_ERaERb_0480*	24.91
Anti-estrogenic (1/13)	OT_ER_ERaERb_0480*	24.91	0
Androgenic (2/10)	NVS_NR_hAR†	11.05	0
	NVS_NR_cAR	19.63
Anti-androgenic (2/8)	NVS_NR_hAR	11.05	0
	NVS_NR_cAR	19.63
Thyroid receptor agonist activity (0/3)	Negative	NA	NA
Thyroid receptor antagonist activity (0/2)	Negative	NA	NA
Hepatic catabolism and thyroid hormone clearance (27/77)	ATG_Ahr_CIS_up	33.88	NA
	ATG_PBREM_CIS_up	16.00
	ATG_PPARg_TRANS_up	31.52
	ATG_PPRE_CIS_up	55.54
	ATG_PXR_TRANS_up	17.79
	ATG_PXRE_CIS_up	7.10
	CLZD_CYP1A1_24	12.03
	CLZD_CYP1A1_48	11.32
	CLZD_CYP1A1_6	8.89
	CLZD_CYP1A2_24	11.94
	CLZD_CYP1A2_48	11.90
	CLZD_CYP2B6_24	8.89
	CLZD_CYP2B6_48	7.73
	CLZD_CYP2B6_6	7.04
	CLZD_CYP3A4_24	5.82
	CLZD_CYP3A4_48	5.67
	CLZD_SULT2A1_48	8.29
	CLZD_UGT1A1_24	9.66
	NVS_ADME_hCYP1A1	2.09
	NVS_ADME_hCYP2C19	1.18
	NVS_ADME_hCYP2C9	7.63
	NVS_ADME_hCYP3A4	4.89
	NVS_ADME_rCYP1A1	16.37
	NVS_ADME_rCYP2B1	0.51
	NVS_ADME_rCYP3A1	8.94
	NVS_NR_hPXR	3.68
	Tox21_AhR	88.51

* This assay may inform E and anti-E models. This assay dose-response is also annotated with caution flag 11: “Borderline active,” as the highest concentration tested which may decrease the reliability of this AC50 value.

† These assays may inform A and anti-A models.

‡ NA indicates there was no AUC model score available for the endocrine hypothesis.

Table 7. Propiconazole: Endocrine-related HTS bioactivity data.

Propiconazole Screening Information Estimated cytotoxicity limit = 4.6 μM
Endocrine activity (positives/total # assays)	Assay	Propiconazole (AC50, uM)	AUC model score
Steroidogenesis (2/2)	NVS_ADME_hCYP19A1	2.24	NA
	Tox21_Aromatase_Inhibition	23.76
Estrogenic (3/16)	ACEA_T47D_80hr_Negative	82.41	0
	ATG_ERa_TRANS_up	26.14
	ATG_ERE_CIS_up	1.65
Anti-estrogenic (2/14)	Tox21_ERa_BLA_Antagonist_ratio	71.27‡	0
	Tox21_ERa_LUC_BG1_Antagonist	56.36
Androgenic (3/9)	NVS_NR_cAR†	12.89	0
	NVS_NR_hAR†	19.88
	OT_AR_ARSRC1_0960†	35.39
Anti-androgenic (5/8)	NVS_NR_cAR†	12.89	0.11
	NVS_NR_hAR†	19.88
	OT_AR_ARSRC1_0960†	35.39
	Tox21_AR_BLA_Antagonist_ratio	33.48
Parallel viability assays	Tox21_AR_BLA_Antagonist_viability	51.75
	Tox21_AR_LUC_MDAKB2_Antagonist	69.99¶
Thyroid Receptor Agonist Activity (0/3)	Negative	NA	NA
Thyroid Receptor Antagonist Activity (1/2)	Tox21_TR_LUC_GH3_Antagonist	54.73	NA
Hepatic catabolism (27/76)	ATG_Ahr_CIS_up	23.04	NA
	ATG_PBREM_CIS_up	48.23
	ATG_PXRE_CIS_up	5.54
	CLZD_ABCB1_24	7.96
	CLZD_ABCB1_48	6.23
	CLZD_ABCG2_48	8.32
	CLZD_CYP1A1_24	11.18
	CLZD_CYP1A1_48	7.36
	CLZD_CYP1A1_6	8.70
	CLZD_CYP1A2_24	5.91
	CLZD_CYP1A2_48	9.18
	CLZD_CYP1A2_6	5.94	NA
	CLZD_CYP2B6_24	0.49
	CLZD_CYP2B6_48	0.46
	CLZD_CYP2B6_6	0.47
	CLZD_CYP3A4_24	0.53
	CLZD_CYP3A4_48	2.96
	CLZD_SLCO1B1_48	0.034
	CLZD_SULT2A1_48	7.44
	NVS_ADME_hCYP1A1	9.01
	NVS_ADME_hCYP2B6	2.63
	NVS_ADME_hCYP2C19	0.57
	NVS_ADME_hCYP2C9	4.41
	NVS_ADME_rCYP2B1	0.38
	NVS_ADME_rCYP3A1	1.95
	NVS_NR_hCAR_Antagonist	32.03
	Tox21_AhR	23.27

† These assays may inform androgenic and anti-androgenic models

‡ The Tox21_ERa_BLA_Antagonist_ratio assay dose-response is annotated with caution flag 12: “Borderline inactive,” decreasing the reliability of this AC50 value.

¶ The Tox21_AR_LUC_MDAKB2_Antagonist assay dose-response curve is annotated with caution flag 6: “Only highest conc above baseline, active,” indicating that only the maximum concentration tested exceeded the range of baseline values.

Table 8. Myclobutanil: endocrine-related HTS bioactivity data.

Myclobutanil Screening Information Estimated cytotoxicity limit = 4.69 μM
Endocrine activity (positives/total # assays)	Assay ID	AC50 (μM)	AUC model score
Steroidogenesis (2/2)	NVS_ADME_hCYP19A1	0.672	NA
	Tox21_Aromatase_Inhibition	5.16
Estrogenic (2/15)	ATG_ERa_TRANS_up	34.67*	0
	ATG_ERE_CIS_up	5.11†
Anti-estrogenic (0/13)	Negative	NA	0
Androgenic (0/8)	Negative	NA	0
Anti-androgenic (0/6)	Negative	NA	0
Thyroid receptor activity (0/4)	Negative	NA	NA
Hepatic catabolism (19/79)	ATG_PXRE_CIS	13.86	NA
	CLZD_CYP1A1_48	6.73
	CLZD_CYP1A2_24	5.99
	CLZD_CYP1A2_48	11.84
	CLZD_CYP2B6_24	11.28
	CLZD_CYP2B6_48	7.26
	CLZD_CYP2B6_6	4.15
	CLZD_CYP2C9_48	3.91
	CLZD_CYP3A4_24	6.07
	CLZD_CYP3A4_48	5.93
	CLZD_CYP3A4_6	5.24
	CLZD_UGT1A1_24	14.60
	NVS_ADME_rCYP3A1	3.69
	NVS_ADME_rCYP2B1	1.52
	NVS_ADME_hCYP2B6	5.66
	NVS_ADME_hCYP1A1	10.66
	NVS_ADME_rCYP3A2	2.77
	NVS_ADME_hCYP2C19	0.58

* The dose–response curve information for ATG_ERE_CIS_up is accompanied by 1 caution flag: 16: “Hit-call potentially confounded by overfitting.”

† The dose–response curve information for ATG_ERa_TRANS_up is accompanied by 3 caution flags that suggest decreased relevance (6: “Only highest conc above baseline, active”; 11: “borderline active; and, 16: “Hit-call potentially confounded by overfitting”).

Table 9. Triadimefon: mammalian EDSP Tier 1 assay results plus published OSRI.

Study	Methods	Concentrations tested	E	Anti-E	A	Anti-A	S	Anti-T	Additionalfindings
ER binding assay OCSPP 890.1250 (Willoughby 2012b)	ER binding in vitro	10^−10 to 10^−4 M	Non-interacting	Non-interacting
AR binding assay (OCSPP 890.1150) (Willoughby 2012a)	AR binding in vitro using rat prostate	10^−11 to 10^−3 M, precipitation at 10^−3			Non-interacting	Non-interacting
ER-TA OCSPP 890.1300 (Willoughby 2012c)	ER transactivation in vitro in HeLa cells	10^−10.3 to 10^−4 M	Activates transcription at 30 – 100 μM by 18–30%
ER-TA and AR-TA. OSRI cited: (Kojima et al. 2004)	CHO cells with hERα or hERβ or hAR and luciferase reporter (± 17β-estradiol or DHT)	10 nM to 10 μM	No Activation	No Inhib	No Activation	No Inhib
Aromatase inhibition Waived; USEPA (2007a) validation study (OCSPP 890.1200)	Aromatase inhib in vitro	10^−4 to 10^−8 M		May have an anti-E effect	May have an A effect		Inhib aromatase with IC50 = 1.71 μM
Aromatase inhibition OSRI cited: (Vingaard et al. 2000)	Aromatase inhib with human placental microsomes	1 – 100 μM		May have an anti-E effect.	May have an A effect.		Inhib aromatase with IC50 = 32 μM
Aromatase inhibition OSRI cited (Ohno et al. 2004)	KGN cells incubated with androstenedione; measured levels of estrone	500 pM to 50 μM		May have an anti-E effect	May have an A effect		Inhibits aromatase with IC50 = 3.59 μM
Aromatase inhibition OSRI cited (Trosken et al. 2004)	Aromatase inhib with human recombinant CYP19	A range covering three orders of magnitude		May have an anti-E effect.	May have an A effect		Inhibits aromatase with IC50 = 17.5 μM
Steroidogenesis Waived (OCSPP 890.1550)	H295R cell line assay	NA
Steroidogenesis, OSRI cited: (Goetz et al. 2009)	H295R cell line	1, 3, 10, 30 and 100 μM	↑ estradiol at 3 μM; no estradiol effect at >3 μM (not dose-responsive)			↓ testosterone by 20–80% at 10 – 100 μM	Data suggest effects on in vitro S
	Testis organ culture	1, 10 and 100 μM				Negative	↓ testosterone and androstenedione production and ↑ 17α-hydroxyprogesterone and progesterone at 10–100 μM (consistent with CYP17A1 inhib)
	In vivo 30 d dietary exposure in ♂ Wistar rats	126 mkd	No change in serum estradiol		↑ serum and intra-testicular testosterone	Minor ↓ in epididymis wt	Dysregulation of A and Anti-A endpoints interpreted to be related to S or systemic toxicity		↓ BW and ↑ liver wt; small ↓ in pituitary wt
Uterotrophic Assay Waived (OCSPP 890.1600)	In vivo uterine wt response to a minimum of 3 d of exposure	NA (OSRI; see Rockett et al. 2006 and multi-generation studies)
Female Pubertal Assay (OCSPP 890.1450) Waived	In vivo pubertal landmarks	NA (OSRI; see Rockett et al. 2006 and multi-generation studies)
Female reproductive development study OSRI cited: (Rockett et al. 2006)	In vivo pubertal landmarks following perinatal and PND (PND22-PND99) exposure	100, 500 and 1800 ppm Depending on maternal age, doses were 7.9–17.8, 35.9–94.1 and 94.3–300.1 mkd Depending on F1 age, doses were 7.3–14.9, 39.1–76.8 and 139–267 mkd	↑ rel ovary wt with no effects on histopath at 1800 ppm in F1; no effects on serum estradiol	Delayed VO at 1800 ppm in F1; disrupted estrous cyclicity at weeks 5 and 6 only at 1800 ppm in F1; no effects on serum estradiol			Effects on E status suggested (see anti-E column) No effect on: serum estradiol levels	Mild follicular cell hypertrophy in T at (1800 ppm) 136 mkd in 2/4 of rats	↓ maternal (gestation and week 1 and –2 lactation) and F1 (weaning to necropsy) feed intake at 1800 ppm; ↑ rel liver wt and ↑ incidence of panlobular hypertrophy at 1800 ppm; ↓ BW for PND99 animals at 1800 ppm
Hershberger Assay (OCSPP 890.1400) (Davis 2011)	Short-term effects on wts of 5 androgen-sensitive organs in rats (± testosterone admin.)	25, 50 and 100 mkd; 10 d post-exposure; agonist and antagonist (with admin of testosterone proprionate, TP)			Agonist mode: Negative. Findings: ↑ ventral prostate wt by 29% at 100 mkd; no effect on glans penis, Cowper’s gland, levator ani plus bulbocavernous muscle complex (LABC) or seminal vesicles.	Antagonist mode: Negative. Findings: co-admin with TP at 100 mkd ↓ BWs; ↓ LABC wt 14%; no effect on glans penis, Cowper’s gland, ventral prostate, seminal vesicle, or liver wt			↓ BWG at 50 and 100 mkd; ↑ abs (20%) and rel liver wt at 100 mkd
Male Pubertal Assay Waived (OCSPP 890.1500)	In vivo pubertal landmarks	NA (OSRI; see Goetz et al. 2007 and multi-generation studies)
OSRI cited: Goetz et al. (2007)*	♂ repro dev landmarks following perinatal and PND 22-92 exposure	100, 500 and 1800 ppm mkd not reported; likely top dose (1800 ppm) ranged 94.3–300.1 mkd (see Rockett et al. 2006)			• ↑ testes abs/rel wt at 500 ppm on PND22, and at 100 and 500 ppm on PND50 (no high dose ♂ available), and ↑ rel testis wt only for 1800 ppm on PND92 in F1• ↑ AGD at 1800 ppm in F1 ♂;• ↑ serum testosterone at 500 and 1800 ppm in F1 ♂; no effect on testes histopath	• Delayed PPS at 1800 ppm in F1 ♂• ↓ mating by F1 ♂ at 1800 ppm with no sperm effects• Failure to inseminate resulted in inability to measure fertility		No effects on T wt or histopath. T4 ↓ at 1,800 ppm on PND 92	• Decreased pup survival (↓47.37%) at 1800 ppm • 20–30% ↓ BW (PND1-92) at 1800 ppm. ↓ rel brain wt (PND92) at 1800 ppm• ↓ feed consumption in F1 ♂for much of the study• ↑ 20% rel liver wt at PND92 with 1800 ppm• No copulatory plugs for any of the F1 1800 ppm matings.
OSRI cited: Tully et al. (2006)	Testis and hepatic biomarkers following a 14-d exposure in ♂rats	0, 5, 50 and 115 mkd			No effect on serum testosterone; no effects on testis wt or histopath. No effect on sperm morphology or motility		No effects on T histopath	Centrilobular hypertrophy and ↑rel liver wt (9–20%) at 50 and 115 mkd
OSRI cited: Wolf et al. (2006)	Markers of T disruption; dietary exposure in ♂Wistar rats for 4, 30, or 90 d	100, 500 and 1800 ppm						• ↓ T4 (4, 30 d exposure), ↓ T3 (4, 30 d exposure,) and ↓ TSH (4 d exposure) with 1800 ppm;• T follicular cell hypertrophy, colloid depletion, and cell prolif. with at 30 d but not 90 d at 1800 ppm
OSRI cited: Hester and Nesnow (2008)	Markers of T disruption; dietary exposure to ♂Wistar rats for 30 or 90 d	1800 ppm in feed (106.7 mkd)						↑ PPARγ, markers of oxidative stress (CYP activation), cell proliferation and migration, and fatty acid biosynthesis inT tissue

* Goetz et al. (2007): rel ventral prostate wt was ↑ by 2–3% at 100 and 500 ppm, but not at 1800 ppm in F1 ♂ at PND92. Rel seminal vesicle wt was ↑ at PND92 at 500 ppm, but not at 1800 ppm. With 1800 ppm exposure, the abs seminal vesicle wt was ↓, but with no effects on rel seminal vesicle wt. Considering the minor level of changes, inconsistent dose-response relationships, and lack of micropathology, we did not consider these findings relevant.

Table 10. Propiconazole: mammalian EDSP Tier 1 assay results plus published OSRI.

Study	Methods	Concentrations tested	E	Anti-E	A	Anti-A	S	Anti-T	Other info., additional findings
ER binding assay OCSPP 890.1250 (Willoughby 2012d, e)	ER binding in vitro using rat uterine cytosol	10^−10 to 10^−3 M	Non-interacting	Non-interacting
AR binding assay (OCSPP 890.1150) Waived	AR binding in vitro using rat prostate	NA
AR binding assay OSRI cited: Bauer et al. (2002)	Recombinant human AR on microtiter plates	10–200 μM			Ki = 95.9 μM	Ki = 95.9 μM
AR binding assay OSRI cited: Kojima et al. (2001)	AR reporter assay in CHO cells (agonist and antagonist assays)	0.01–10 μM.			Non-interacting	6.2 μM =  20% inhib
AR Binding Assay OSRI cited: Vinggaard et al. (2008).	Human AR reporter assay in CHO cells (antagonist assay)	1, 3, 10 and 30 μM				3 μM < IC25 < 10 μM
AR Binding Assay OSRI cited: Kjaerstad et al. (2007)	Human AR reporter assay in CHO cells (antagonist assay)	0.025–50 μM				LOEC = 25 μM
ER-TA OCSPP 890.1300 Waived	ER transactivation in vitro in HeLa cells	NA
ER-TA. OSRI cited: (Kjaerstad et al. 2007)	Cell proliferation in MCF-7 cells by ELISA	1 nM to 150 μM	AC50 > 25 μM	IC50 = 52 uM
ER-TA. OSRI cited: (Kojima et al. 2004)	CHO cells with ERα or ERβ and luciferase reporter (± estradiol)	10 nM to 10 μM	No activation	No inhibition
ER-TA. OSRI cited: (Hurst and Sheehan, 2003)	yeast cells with hERα and Lac-Z reporter	Inappropriate test system (yeast) for a fungicide	Not applicable	Not applicable
Aromatase inhibition Waived; (OCSPP 890.1200)	human recombinant CYP19 incubated with ^3H-androstendione	NA
Aromatase inhibition OSRI cited: (Ohno et al. 2004)	KGN cells incubated with androstenedione; measured levels of estrone	100 pM–10 μM		May have an anti-E effect	May have an A effect		Inhibits aromatase with IC50 =  0.968 μM
Aromatase inhibition OSRI cited: (five other studies)	Aromatase inhib assay; Various methods	Multiple; up to 100 μM		May have an anti-E effect	May have an A effect		IC50 values = 3.2–8.25 μM		Laville et al. (2006), Vingaard et al. (2000), Trosken et al. (2004, 2006b), Sanderson et al. (2002)
Steroidogenesis Waived (OCSPP 890.1550)	H295R cell line assay, measure 17β-estradiol and testosterone content	NA
Steroidogenesis OSRI cited: Goetz et al. (2009)	H295R cell line assay, measure 17β-estradiol and testosterone	1–100 μM		Possible anti-E effect at high concentrations		Possible anti-A effect at high concentrations	↑ Estradiol at 1–3 μM ↓ Estradiol, testosterone and progesterone at ≥10 μM
Steroidogenesis OSRI cited: Kjaerstad et al. (2007)	H295R cell line assay, measure 17β-estradiol and testosterone	0.1–30 μM		Possible anti-E effect at high concentrations		Possible anti-A effect at high concentrations	↓ Estradiol, testosterone at ≥1 μM ↑ Progesterone at ≥1 μM
Uterotrophic assay (OCSPP 890.1600) Coder (2012)	In vivo uterine wt response to a minimum of 3 d of exposure	0, 175, 400 and 500 mkd in OVX rats	Negative
Female pubertal assay (OCSPP 890.1450) Waived	In vivo pubertal landmarks	NA
Female reproductive development study; female pubertal assay OSRI cited: (Rockett et al. 2006)	In vivo pubertal landmarks following perinatal and PND (GD6-PND99) exposure	0, 100, 500 and 2500 ppm in diet	No effects that would indicate an E effect, including• AGD in ♀• Litter size• Gender ratio• Age at VO• Estrous cycling• Endo-crine sensitive organs wts• Histology• Serum estradiol	No effects that would indicated an anti-E effect (see prior column)			No effects on serum estradiol levels	No effects that would indicate an anti-T effect:• T wt• T micropath
Hershberger Assay (OCSPP 890.1400) Waived	Short-term effects on wts of 5 androgen-sensitive organs in rats (± testosterone admin)	NA
Hershberger Assay OSRI cited: Kjaerstad et al. (2007) and Taxvig et al. (2008)	Anti-A Hershberger assay; castrated ♂ rats treated with testosterone + increasing doses of propiconazole	50, 100 and 150 mkd, orally for 7 d			Agonist mode: Negative (see Goetz et al. 2007 below, under male pubertal assay)	Antagonist mode: Negative. Findings: No effect on wt of prostate, seminal vesicles, LABC, bulbourethral gland, pituitary		No effect on T4 levels or T wt	No effect on LH levels; ↑ FSH at 150 mkd
Hershberger Assay OSRI cited: Taxvig et al. (2008)	2nd study: pregnant rats treated GD7–GD21	50 mkd	No effect on implantations or AGD in fetuses	No effect on implantations or AGD in fetuses	No effect on implantations or AGD in fetuses	No effect on implantations or AGD in fetuses	Negative. No effect on testosterone, progesterone or estradiol in dams; No effect on hormones in tissues of fetuses (estradiol, testosterone, progesterone)		↑ 17α-hydroxy-progesterone in serum of dams
Male pubertal assay Waived (OCSPP 890.1500)	In vivo pubertal landmarks	NA
Male Pubertal Assay OSRI cited: Goetz et al. (2007)*^b	♂ repro dev landmarks following perinatal and PND (GD6 – PND92) exposure to Wistar rats	0, 100, 500 and 2500 ppm in diet			↑ AGD at 2500 ppm in F1 ♂;. no effect in ♀ or in several other similar studies (Taxvig et al. 2008)	Negative. No effects on: age at PPS, wt of prostate, seminal vesicles, epididymis, testis wt*; sperm parameters; micropathology of androgen-sensitive organs	Negative. No consistent effects on serum E levels or testosterone† in F1 ♂	No effects on T wt, histopath or T3, T4 and TSH levels in F1 ♂, PND 92	No effects on repro success of treated F1 ♂ paired with untreated ♀ (any dose level)
Male Pubertal Assay OSRI cited: Tully et al. (2006)	Testis and hepatic biomarkers following a 14-d exposure in ♂ SD rats	0, 10, 75 and 150 mkd			No effects on prostate, seminal vesicle, or testis wts. No effects on histopath, sperm morphology or sperm motility.	No effects on prostate, seminal vesicle, or testis wts. No effects on histopath, sperm morphology or sperm motility.	Negative. No effects on serum estradiol or testosterone, or on LH or FSH levels.	No effects on T histopath	↑ liver wts (13–19%), 75and 150 mkd; hepatocyte hypertrophy
Thyroid-Related Tests in Mammals OSRI cited, supplemental to OCSPP 890.1100: Wolf et al. 2006	Markers of T disruption; dietary exposure in ♂ Wistar rats for 4, 30, or 90d	0, 100, 500 and 2500 ppm						↓ serum T4 on ds 4 and 30 (2500 ppm), but T4, T3 and TSH normal by d 90. No effect on T histology	↑ UDPGT activity in liver (2500 ppm)

* Goetz et al. (2007): Testis wt was determined at PND1, 22, 50 and 92, and was ↑ only on PND22 at 2500 ppm (rel wt alone) and on PND50 at 500 ppm (abs, rel wt) and 2500 ppm (rel wt alone), but not at any other intervals. Considering the lack of consistency in this study and across other studies, plus the changes in relative wt secondary to lower BW, this was not considered evidence of a biologically significant change.

† Goetz et al. (2007): Testosterone levels in F1 ♂ were ↑ on PND92 at 500 and 2500 ppm, but not on PND50 or in other studies. Considering the lack of consistency, this was not considered evidence of a biologically significant change.

Table 11. Myclobutanil: mammalian EDSP Tier 1 assay results plus published OSRI.

Study	Methods	Concentrations tested	E	Anti-E	A			Anti-A	S	Anti-T	Additional findings
ER Binding Assay OCSPP 890.1250 (LeBaron et al. 2011a)	ER binding in vitro	10^−10 to 10^−3 M	Non-interacting	Non-interacting
AR Binding Assay (OCSPP 890.1150) (LeBaron et al. 2011b),	AR binding in vitro using rat prostate	10^−10 to 10^−3 M			Equivocal at 1mM			Equivocal at 1mM
ER-TA OCSPP 890.1300 (LeBaron et al. 2011c)	ER transactivation in vitro in HeLa cells	10^−10 to 10^−4 M	No activation
ER-TA. OSRI cited: (Hurst & Sheehan 2003)	Yeast cells with hERα and Lac-Z reporter	Inappropriate test system (yeast) for a fungicide.	Not applicable	Not applicable
Aromatase inhibition (OCSPP 890.1200) (Coady & Sosinski 2011)	Aromatase inhib in vitro	10^−10 to 10^−8 M		May have an anti-E effect	May have an A effect				Inhib aromatase with IC50=0.1 μM
Aromatase inhibition OSRI cited: (Trosken et al. 2004)	A fluorimetric assay based on human recombinant CYP19 enzyme with dibenzylfluorescein (DBF) as a substrate was used to compare the inhibitory potency	A range covering 3 orders of magnitude		May have an anti-E effect	May have an A effect				Inhib aromatase with IC50=0.47 μM
Aromatase inhibition OSRI cited: (Trosken et al. 2006b)	Microsomes (“Supersomes”) containing human aromatase (CYP19) coexpressed with human cytochrome P450 reductase (baculovirus/insect cell expressed) were from BD Gentest. Testosterone was utilized as the substrate	Not provided		May have an anti-E effect	May have an A effect				Inhib aromatase with IC50 = 47μM
Steroidogenesis (OCSPP 890.1550) (LeBaron et al. 2011d)	H295R cell line assay	10^−10 to 10^−4 M		↓estradiol at the three highest concentrations tested (1, 10 and 100 μM).				↓testosterone production only at the assay limit concentration (100μM).	Data suggest effects on in vitro S (≥1 μM)
Steroidogenesis, OSRI cited: (Goetz et al. 2009)	H295R cell line	1, 3, 10, 30 and 100 μM		↓ estradiol ≥3 μM				↓ testosterone ≥ 1μM	Data suggest effects on in vitro S (≥1 μM)
Uterotrophic Assay (OCSPP 890.1600) (Marty & Brooks 2011)	In vivo uterine wt response to a minimum of 3 d of exposure	0, 50, 150 and 475 mkd	Negative
Female Pubertal Assay (OCSPP 890.1450) (Marty et al. 2011a)	In vivo pubertal landmarks	0, 50, 200 and 400 mkd	• Negative• No effects that would indicate an E effect, including• Age/BW at VO• Estrous cycling, Pituitary, ovarian or uterine wts• Histology• Serum estradiol	Negative. No effect that would indicate an anti-E effect (see prior column)					Negative. No effect that would indicate altered estrogen synthesis (see “estrogenic” endpoints); also no effect on: adrenal wts	Negative. No effects that would indicate an anti-T effect:• T wt• T micropath• T4 or TSH levels	Liver:• ↑ Abs/rel wts at 200 and 400 mkd (18–41%)• Increased alanine amino-transferase and gamma glutamyl trans-peptidase at 400 mkd• Slight hepatocellular hypertrophy and ↑ mitotic figures at 200 and 400 mkd
Female reproductive development study OSRI cited: (Rockett et al. 2006)	In vivo pubertal landmarks following perinatal and PND (GD6-PND99) exposure	0, 100, 500 and 2000 ppm in diet (mkd ranges: 0, 8–19.1, 38.7–-93.8 and 141.3–347.3)	Negative. No effects on• Serum E levels• Estrous cycling• Uterine wt• Ovarian histopath	No effects on:• Serum estradiol levels• Estrous cycling• Uterine wt• Ovarian histopath Effects:• ↑AGD and delayed VO (+2.2 ds)in ♀ pups and ↑ovarian wt (45%) at 2000 ppm					Effects on E status suggested (see anti-Ecolumn) No effect on: serum estradiol levels	No effects that would indicate an anti-T effect:• T wt• T histopath	• ↓ feed intake (wk 2 lactation) at 2000 ppm• At PND 0 and VO, there were no significant differences in BW• No effects on rel brain hypo-thalamus and liver wts
Hershberger Assay (OCSPP 890.1400) (Marty & Marshall 2011)	Short-term effects on wts of 5 androgen-sensitive organs in rats (± testosterone admin.)	0,25,125 and 500 mkd; 10 d post-exposure; agonist and antagonist (with admin of testosterone proprionate)			Agonist mode:Negative.No effectson LABC, glanspenis, seminalvesicle, ventralprostate orCowper’sgland			Antagonist mode: Negative. No effects on glans penis, Cowper’s gland, ventral prostate, or seminal vesicle wts. ↓ only in LABC wt (20%) at 500 mkd			• ↓ BWG (62–81%) (TD1-4) at 500 mkd with or without testosterone propionate Liver:• ↑ Abs wts at 125 (20%) and 500 mkd (53–59%)• ↑ histopath (hepato-cellular hypertrophy, focal/multi-focal hepato-cellular necrosis)
Male Pubertal Assay (OCSPP 890.1500) (Marty et al. 2011b)	In vivo pubertal landmarks	0, 50, 200 and 400 mkd			No effectson A endpointsconsistentwithandrogenicity			No effects on:• testicular or epididymal histopath Effects on: • Slight delay in PPS (+1.7 ds) at 400 mkd• Decreased serum testosterone (49 or 85%)• Decreased abs pituitary wts at all dose levels• Decreased abs. seminal vesicle and ventral prostate wts at 200 and 400 mkd• Decreased abs. dorso-lateral prostate and LABC at 400 mkd	Increased rel. adrenal wts at 400 mkd (see anti-A effects in prior column)	No effects on T wt or on T4 levels ↑ serum TSH (58%) and altered T histopath at 400 mkd (↑ follicular cell height and ↓ colloid)	• ↓ BW (6.6 or 6.2%) and BWG (8.3 or 8.2%) at 200 or 400 mkd Liver:• ↑Abs/rel wts (12.5–21.3%) at 200 and 26.7–36.5% at 400 mkd along with hypertrophy • ↑gamma glutamyl trans-peptidase at 400 mkd
OSRI cited: Goetz et al. (2007)	♂ repro dev landmarks following perinatal and PND (GD6 – PND92) exposure to Wistar rats	0,100,500 and 2000 ppm in diet (mkd range for dams during gestation and lactation: 0, 8–19.1, 38.7–93.8 and 141.3–347.3; mkd ranges during postweaning period: 0, 6.1–15.8, 32.9–77.2 and 133.9–280.2)			No effects:• Age at PPS• Serum estradiol or LH levels• Sperm morphology/motility,wt of seminal vesicles,epididymis,hypothalamus,brain or hippocampus• Histopath ofandrogen-sensitiveorgans (testis,epididymis, ventralprostate, pituitary)• Total numberof implantationsites, live ordead fetuses, liveor dead embryos,number of resorptionsor postimplantationloss% fertility(no. of livefetuses/no.CLs in successfulpregnancies) Effects:*• ↑ AGD in ♂ at 2000 ppm• ↓ rel pituitary wt at PND92 at 500 and 2000 ppm↑ abs testes wt at PND1 at 100 and 2000 ppm, andat PND 22 at 500 ppm with nochanges at PND 50 or92 at any dose level• ↑abs and rel ventralprostate wt at PND 92at 500 ppm (no dose-response)• Impaired insemination withuntreated ♀ (reducedratio of vaginal smearswith sperm present)at 2000 ppm• ↓fertility (↓ratioof successfulpregnancies permated, untreated ♀)at 500 and 2000• ↑serum testosteroneat PND 92/99 at500 and 2000 ppm					No effects that would indicate an anti-T effect:• T wt• T micropath• Serum T3, T4 and TSH	↓ BW (12%) and feed intake in postweaning ♂ at 2000 ppm• ↓ BW at PPS 7.8% at 2000 ppm• ↓ survival rates (82.6%) in litters exposed to 2000 ppm during gestation Liver:• ↑ Rel wt at PND 1, 50 and 92 at 2000 ppm (9–13%)• Centri-lobular hepato-cellular hyper-trophyat PND 92 at 2000 ppm
OSRI cited: Tully et al. (2006)	Testis and hepatic biomarkers following a 14-d exposure in ♂ rats	0, 10, 75 and150 mkd for 14 d				• No effect on testis wts• ↑serum testosterone• ↓ sperm motility withno effect on testis histopath				No effects onT histopath	Liver: ↑ Rel wts at 75 and 150 mkd (6–8%) with centrilobular hypertrophy
OSRI cited: Wolf et l. (2006)	Markers of T disruption; dietary exposure in ♂ Wistar rats for 4, 30, or 90d	100, 500 and 2000 ppm								No effect on T histology or TSH at any time or exposure• ↓ T4 only (4 d exposure) and T3 only (30 d exposure) (2000 ppm), but• T4, T3 not affected after 90 d	Liver:  ↑ UDPGT activity in liver (2000 ppm)

* Goetz et al. (2007): Testis wt determined at PND1, 22, 50 and 92 were inconsistent in myclobutanil treated ♂ with ↑ testes abs wt at PND 1 at 100 and 2000 ppm and at PND 22 only at 500 ppm. ↑ rel testes wt on PND1 at 500 ppm and on PNDs 22 and 50 at 2000 ppm. No wt changes (both rel and abs) were seen on PND 92 at any dose level. Considering the lack of consistency in this study and across other studies, this was not considered evidence of a biologically-significant change. Similarly rel ventral prostate wt was ↑ at 500 ppm, but not at 2000 ppm in F1 ♂ at PND92. Considering the lack of consistency in this study and across other studies, this was not considered evidence of a biologically-significant change. In addition, testosterone levels in F1 ♂ were ↑ on PND92 at 2000 ppm, but not on any other time points or dose levels or in other studies. Considering the lack of consistency, this was not considered evidence of a biologically-significant change.

Table 12. Triadimefon: Part 158 Guideline Toxicology Studies.

Study	Concentrations tested	E	Anti-E	A	Anti-A	Anti-T	Additional findings
Subchronic oral rat (Mohr 1976)	0, 50, 200, 800 and 2000 ppm (0, 2.5, 10, 40 and 100 mkd)	No effects (ovary wt or histopath)		• ↑ rel testes wt (19%) at 2000 ppm• No effects on testes histopath.	No effects on testes histopath	33% ↑ rel T wt, ♀ rats only at 2000 ppm	↑ rel liver wt at ≥50 ppm in ♀ (26–73% at 2000 ppm) and ≥200 ppm in ♂ (27–36%, non-dose dependent)
Subchronic oral dog (Hoffman and Luckhaus 1974)	0, 150, 600 and 2400 ppm (0, 3.75, 15 and 60 mkd)	No effects (ovary or uterine wt or histopath)		No effects (prostate or testes wt, prostate,epididymis, and testes histopath)		No effects (T wt or histopath)	• ↑ rel liver wt (16%) and ↓ BW and food consumption at 2400 ppm• ↑ hepatic enzymes at ≥600 ppm
DNT (Sheets et al. 2008)	0, 100, 300 and 800 ppm (0, 8.0, 23.9 and 71.3 mkd)	No effects (age or BW at VO)		No effects (age or BW at PPS)		No effects (eye opening, brain wt and volume, motor activity, and auditory function (no loss of startle response) at ≤800 ppm	• ↓ maternal BWG (5%) on GD13 and LD0• ↑ incidence of deviated snout, ↑ startle amplitude on PND60 and number of trials to criterion during retention phase of passive avoidance test (♀ only)• ↓ ♂ pup BWG prior to weaning at 300 ppm• Maternal and offspring NOAEL =300 ppm
Chronic rat (Bomhard and Schilde 1991)	0, 50, 300 and 1800 ppm (approx. 2.5, 15 and 90 mkd)	No effects (ovary or uterine histopath; no organ wt available)		↑ Rel testes wt (5%) at 1800 ppm at 104 wk	No effects (testes or seminal vesicles histopath)	Slight ↑ in T cystic hyperplasia and follicular cell adenomas at 1800 ppm (2 ♀/50 and 4 ♂/50, within historical control range)	• ↑ rel and abs liver wt at 300 ppm (8%) and 1800 ppm (32%) in ♀, and at ↑ rel liver wt (5%) in ♂ at 1800 ppm• ↓ ♂ (7%) and ♀ (13%) BW at 1800 ppm (effects at 104 wk)
Mouse oncogenicity (Bomhard 1986)	0, 50, 300 and 1800 ppm (approx. 7.1, 43.0 and 257 mkd)	No effects (ovary or uterine histopath, no wt available)		No effects (testes wt, or on testes or seminal vesicles histopath)		No effects on T histopath, no wt available)	• ↓ BWG in ♂ and ♀ (≤ 20%) at 1800 ppm• ↑ nonneoplastic (centrilobular hypertrophy) changes and hepatocellular tumors at 1800 ppm• ↑ liver wt (50–60%) at 1800 ppm in ♂ and ♀
Chronic dog (Hoffman and Groning 1978)	0, 100, 330 and 1000 ppm (wk 1–54), or 2000 ppm (wk 55–104) F: 0, 3.5, 12.0, 33.7 (wk 1–54) or 60.4 (wk 55–104) mkd; M: 0, 3.0, 11.4, 34.7 (wk 1–54) or 68.8 (wk 55–104) mkd	No effects (ovary wt or ovary and uterine histopath)		No effects (prostate or testes wt, or on prostate, epididymis and testes histopath)		No effects (T wt or histopath)	↑ rel liver wt and microsomal enzyme induction at 1000 ppm
Rat developmental toxicity (Nagumo et al. 1981; Unger et al. 1982)	Nagumo (1981): 0, 10, 25, 50 and 100 mkd by gavage Unger, 1982: 0, 10, 30 and 90 mkd	No effects on endpoints that would suggest an E or anti-E effect, including• Gestation duration• Sex ratio• Dev abnormalities suggestive of endocrine effects, e.g., urogenital malformations		No effects on endpoints that would suggest an A or anti-A effect, including:• Gestation duration• Sex ratio• Dev abnormalities suggestive of endocrine effects, e.g., urogenital malformations			• ↑ incidence of supernumerary ribs at 50 and 100 mkd• ↓BWG early in gestation at 50–100 mkd• ↑ motor activity at 25–100 mkd• NOAEL range was 10–30 mkd based on maternal BW
Rabbit developmental toxicity (Clemens 1990)	0, 20, 50 and 120 mkd	No effects on endpoints that would suggest an E or anti-E effect, including:• Gestation duration• Sex ratio Dev abnormalities suggestive of endocrine effects, e.g., urogenital malformations		No effects on endpoints that would suggest an A or anti-A effect, including• Gestation duration• Sex ratioDev abnormalities suggestive of endocrine effects, e.g., urogenital malformations			• ↓ fetal wt at 120 mkd and minor delays in ossification at 50–120 mkd• ↓ maternal food consumption and reduced BW early in gestation at 120 mkd• Dev NOEL =20 mkd
Multigenerational rat study (Loser & Lorke 1979)	0, 50, 300 and 1800 ppm (variable mkd dose dependent on lifestage but average exposure 0, 2.5, 15 and 90 mkd)	No effects (ovary histopath at ≤300 ppm; only F3b pups examined at 0, 50 and 300 ppm; no pups available at 1800 ppm)		No effect on testes or epididymis histopath≤ 300 ppm (only F3b pups examined at 0, 50, and 300 ppm; no pups available at 1800 ppm)	↓ mating by F1 ♂ at 1800 ppm	No effect (T histopath at ≤300 ppm; only F3b pups examined at 0, 50, and 300 ppm; no pups available at 1800 ppm)	• ↓ BWG at ≥300 ppm• LOAEL =300 ppm
Multigenerational rat study (Eiben 1984)	0, 50 and 1800 ppm (variable mkd dose dependent on lifestage but average exposure 0, 2.5 and 90 mkd)	• ↑ rel and abs ovary wt at 1800 ppm in P only (no effect in F1)• No effects on ovary or uterus histopath at 1800 ppm (only control and 1800 ppm F1 adults examined)	No effects• 1800 ppm dams mated with control ♂ expressed normal fecundity	• No effect on prostate, seminal vesicles, epididymis, or testes histopath at 1800 ppm (only control and 1800 ppm F1 parents examined)• ↑ testes rel wt by 18% at 1800 ppm in the F1• ↑ serum testosterone by 100% at 1800 ppm (F1 ♂ assessed at 8 mo old and end of study)• No effect on FSH	• ↓ mating (behavior) by F1 ♂ at 1800 ppm with no sperm effects• F0 mating was unaffected	No T wt or histopath available	• ↓ F0 and F1 parental and F1 and F2 pup BW at 1800 ppm• ↑ rel liver wt at 1800 ppm for F0 ♂ and ♀ and F1 ♂ (25%) (↓ for F1 ♀)• For F1–F2 offspring: ↓ litter wt (12%), ↓ viability (50–60%), and ↓ lactation survival (20%) at 1800 ppm• 4 F1 ♂ did not mate at all demonstrated the greatest growth retardation

Table 13. Propiconazole: Part 158 Guideline Toxicology Studies.

Study	Concentrations tested	E	Anti-E	A	Anti-A	Anti-T	Additional findings§
Subchronic rat (Sachsse et al. 1979a)	0, 240, 1200 and 6000 ppm	No effects on ovary wt or histopath. No effect on mammary or uterine histopath		No effects on testes wts; or on prostate, epididymis, and testes histopath		No effects on T histopath	↑ rel ovary and testis wt at 6000 ppm attributed to large BWG ↓ (≥25%)
Subchronic dog (Sachsse et al. 1979b)	0, 50, 250 and 1250 ppm	No effects on ovary wt or histopath. No effect on mammary or uterine histopath		No effects on testes wts; or on prostate, epididymis, pituitary and testes histopath		No effects on T wt or histopath
Chronic rat (Hunter et al. 1982a)	0, 100, 500 and 2500 ppm	No effects on ovary wt or histopath. No effects on mammary, uterus, cervix or pituitary histopath*		No effects on testes wts; or on prostate, epididymis, seminal vesicles, pituitary and testes histopath		No effects on T wt or histopath
Mouse oncogenicity (Hunter et al. 1982b)	0, 100, 500 and 2500 ppm	No effects on ovary wt or histopath. No effects on mammary, uterus or pituitary histopath		No effects on pituitary or testes wts; or on pituitary, prostate, epididymis, and testes histopath		No effects on T wt or histopath
1 year dog (Johnson & Thompson 1985)	0, 5, 50 and 250 ppm	No effects on ovary or pituitary wt or histopath. No effects on uterus, vagina or mammary histopath		No effects on testes wts; or on prostate, epididymis, and testes histopath. A decrease in rel pituitary wt was seen in 250 ppm ♂, but there were no pituitary histology findings†		No effects on T wt or histopath
Rat developmental toxicity (Fritz 1984a; Giknis 1987; Mallow et al. 1987)	30 to 300 mkd	No effects on endpoints that would suggest an E or anti-E effect, including• Gestation duration• Sex ratio• Dev abnormalities suggestive of endocrine effects (e.g., urogenital malformations)		No effects on endpoints that would suggest an A or anti-A effect, including• Gestation duration• Sex ratio• Dev abnormalities suggestive of endocrine effects (e.g., urogenital malformations)			General maternal toxicity (including ↓ BW) at 300 mkd
Rabbit developmental toxicity (Fritz 1984b; Raab 1987)	Fritz (1984b): 0, 30, 90 and 180 mkd Raab (1987): 0, 100, 250 and 400 mkd	No effects that would indicate an E or anti-E effect Slightly ↑ abortion/early parturition at high dose in one study (Raab 1987). No similar findings in second rabbit study (Fritz 1984b)‡		No effects on endpoints that would suggest an A or anti-A effect, including:• Gestation duration• Sex ratio• Developmental abnormalities suggestive of endocrine effects (e.g., urogenital malformations)			Raab (1987): ↓ BW and food consumption at 250 and 400 mkd and clinical signs at 400 mkd
Multigeneration Reproduction Study (Borders & Salamon 1985)	0, 100, 500 and 2500 ppm (0, 8.1, 41.4 and 200.4 mkd – F1 ♀ at wk 10)	No effects on endpoints that would suggest an E or anti-E effect, including• Repro• Sex ratio• Ovary wt• Histology of ovary, uterus, vagina and pituitary		No effects on endpoints that would suggest an A or anti-A effect, including:• Repro• Sex ratio• Testis wt¶, Histology of prostate, seminal vesicles, pituitary and testes with epididymides		No effect on T histopath	At 2500 ppm, ↓ number of pups delivered and surviving to PND4 in F2a litters; ↓ number of pups surviving PND 7-21in F2b litters = offspring toxicity. No evidence of a repro or endocrine-mediated effect

NT, not tested.

* Chronic rat study: US EPA considered incidence of uterus luminal dilatation at 2500 ppm (17/65 versus 4/58 control) to be treatment related, but subsequent examination of the report indicated that this was a normal age-related finding in 2-year old rats, that was recorded under a variety of slightly different pathology descriptors in the control group.

† One-year dog study: in the absence of any micropathology findings or effects on abs organ wt, lower rel pituitary wt (♂ dogs, 250 ppm) was not considered a treatment-related effect.

‡ Rabbit developmental toxicity study (Raab 1987): slight ↑ abortion/early parturition at 400 mkd was seen in the presence of significant maternal toxicity (BW, food consumption, clinical signs) and is considered a result of general toxicity.

¶ Multigeneration repro study: abs testes wts were lower than control at 2500 ppm in F2A and F2B offspring, but not adverse since no effect on rel wts and no micropathology changes. Absolute testis wts in the F1A and F1B offspring were unaffected.

§ In all rat and mouse studies, effects on ↓ BWG and liver wt/liver histopathology were observed that defined the LOAEL and NOAEL values. Liver histopathology in rodents included hepatocyte hypertrophy, vacuolation, necrosis (mice only) and adenomas/carcinomas (mice only; 2500 ppm). NOAEL values for these effects were at 100 ppm (10 mkd) in mice and at 500 ppm (18 mkd) in rats (EPA 2014b).

Table 14. Myclobutanil: Part 158 Guideline Toxicology Studies.

Study	Concentrations tested	E	Anti-E	A	Anti-A	Anti-S	Anti-T	Additional findings – systemic toxicity
Subchronic Mice, 1986 (Goldman et al. 1986a)	Dose levels: 0, 3, 10, 30, 100, 300, 1000, 3000 and 10000 ppm For ♂, 0.40, 1.54, 4.79, 14.1, 42.7, 132, 542 and 2035 mkd and for ♀ 0.62, 2.11, 6.94, 22.9, 65.5, 232, 710 and 2027 mkd	• No effects	• Immature uterus and absence of corpora lutea in the ovaries (no ovulation) at 10000 ppm	• Increased rel testis wt (15%) at 10000 ppm secondary to terminal BW decreases	• No effects	• Adrenals:↑ rel wts at 10,000 ppm secondary to ↓ BW and ↑ eosinophilia and/or hypertrophy of zona fasciculata at ≥1000 ppm	No effects	Systemic toxicity at 3000 ppm (exceeded the MTD at 10,000 ppm)• Severe BW ↓ (21%) at 10,000 ppm with ↓ feed consumption; also ↓ BW in ♂ at 3000 ppm (7%)• Lymphoid necrosis in spleens at ≥3000 ppm and thymus and mesenteric lymph nodes at 10,000 ppm Liver:• ↑ Rel wt at ≥1000 ppm (35–151% in ♂; 18–141% in ♀)• Hepatocellular hypertrophy, vacuolation, necrosis and necrotic hepatitis at ≥1000 ppm
Subchronic Rats, 1984 (O’Hara & DiDonato 1984)	0, 10, 30, 100, 300, 1000, 3000, 10,000 and 30,000 ppm from wk 5 onward (dose levels were ↑) (mkd ranges: 0, 0.52–0.67, 1.6–2.0, 5.2–6.8 15–20, 51.5–65.8 158–195, 585–665 and 1730–1811 mkd)	• No effects	• No effects	• No effects	• No effects	• Vacuolation of adrenal cortex in ♂ at (1000), 3000 and 10,000 ppm and in ♀ at 3000 and 10,000 ppm	• ↑ Rel T wt in ♂ of 3000 (21%) and 10,000 (38%) ppm and in ♀ of the 10000-ppm (19%) group• ↑ small follicle number in T of ♂ at 3000 ppm and 10000 ppm	Systemic toxicity at 10,000 ppm (Exceeded the MTD at 30,000 ppm-):• All were dead at 30000 ppm, including ♂ by TD63 and ♀ by TD49 (exceeded the MTD). ↓ BW at 10000 ppm indicative of systemic toxicity (30% in ♂ and 12% in ♀) Liver:• ↑ Rel liver wts at ≥3000 ppm (38–128% in ♂ and 33–104% in ♀),• Hypertrophy and single cell necrosis at ≥3000 ppm• ↑ BUN, cholesterol and globulin at ≥3000 ppm
Subchronic rats, 1987 (Shimizu 1987)	0, 100, 300 and 3000 ppm; average mkd: 0, 6.2, 18.8 and 191.5 in ♂ and 6.9, 19.6 and 224.9 in ♀; 10/sex/dose	• No effects	• ↑ Abs./rel. ovarian wt only at mid dose 300 ppm (no dose-response)• No histopath changes observed	• No effects	• No effects on pituitary wt• No effects on repro/ASG histopath• ↓ Abs./rel. prostate wt at 100 and 3000 ppm (not dose-related)• Atrophy of seminiferous tubule, giant cell-like change and absence of sperm cells in epididymis in 1 high-dose ♂• These effects are not dose dependent and are considered spontaneous	• ↓ Abs. adrenal wts in ♂ at 3000 ppm; ↓ rel. adrenal wts ≥100 ppm in ♂• Slight vacuolation of cortical cells, slight atrophy of the zona fasciculata, fine vacuolation of zona glomerulosa at high dose	• No effects	Systemic toxicity at 3000 ppm (Exceeded MTD in ♀-: 17% ↓ in BWG) ↓9% in ♂ BWG Liver:• ↑ Abs (9–17%)/rel (19–23%) liver wts at 3000 ppm in males and females Slight or moderate hepatocellular hypertrophy at 3000 ppm with focal necrosis• ↓ Triglycerides and glucose in ♂; ↓ bilirubin in both sexes at 3000 ppm Kidney:• ↑ Abs/rel kidney wts in ♂ at 3000 ppm• Slight vacuolar degeneration of renal tubule epithelium, dilated renal pelvis at 3000 ppm
Subchronic dogs, 1984 (4/sex/group) (McLaughlin & DiDonato 1984)	Dose levels= 0, 10, 200, 800 and 1600 ppm 0, 0.34–0.42, 7.26–7.88, 29.13–32.43 and 56.8–57.97 mkd	• No effects	• ↑ rel ovary wts ≥800 ppm attributed to estrus; not treatment related• No histopath changes observed.	No effects	No effects	• ↑ rel adrenal wts in ♀ at 1600 ppm	No effects	Liver:• ↑ Rel wts at ≥800 ppm (24–41% in ♂ and 12–31% in ♀)• ↑ serum alkaline phosphatase• hypertrophy in both sexes at >200 ppm
Chronic rat, 1986 (110/sex/dose) (Shellenberger & Billups 1986)	Dose levels: 0, 50, 200 and 800 ppm 0, 2.49–3.23, 9.84–12.86 and 39.21–52.34 mkd	• No effects	• ↑ abs and rel ovary wts at 800 ppm (not statistically significant and at 12 mos. only)• No histopath changes observed	• No effects	• ↑ incidence of testicular atrophy (degeneration of seminiferous tubules) at ≥200 ppm)• ↓ testis wt at ≥200 ppm	• No effects	No effects	• ↓ BW (2–9%) in 800 ppm ♂ and ♀ at some intervals up to 96 wks Liver: ↑ rel wts (9–15%) at 800 ppm in both sexes
Chronic rat, 1993 (Wolfe 1993)	Dose levels =0 and 2500 ppm 0 and 106–136 mkd	No effects	No effects	• No effects	Aspermato-genesis, hypospermia and cellular debris in the epididymides at 2500 ppm	No effects	No effects	Liver: Hypertrophy and vacuolation at 2500 ppm
Mouse oncogenicity, 1986 (Goldman and Harris 1986)	Dose levels =0, 20, 100 and 500 ppm 0, 2.7–3.2, 13.7–16.5 and 70.2–85.2 mkd	No effects	No effects	No effects	No effects	No effects	No effects	• Slight/individual necrosis and foci of altered hepatocytes and multifocal hepatocellular vacuolation in ♂ and ♀ at 500 ppm
Mouse oncogenicity, 1993 (targeted study) (Andersen et al. 1993)	Dose levels =0 and 20 00 ppm 0 and 393.5 mkd (only ♀)	No effects	No effects	No effects	No effects	• Adrenal cortex hypertrophy of zona fasciculata at 2000 ppm	No effects	• ↓ BW (12%) and food consumption Liver: ↑ Rel wt (33%), ↑ hepatocellular hypertrophy and vacuolation; single cell necrosis and pigmented Kupfer cells
1 year dog, 1986 (6/sex/group) (Goldman et al. 1986b)	Dose levels =0, 10, 100, 400 and 1600 ppm 0, 0.34–0.40, 3.09–3.83, 14.28–15.68 and 54.22–58.20 mkd	No effects	No effects	No effects	No effects	No effects	No effects	Liver: • ↑ Abs/Rel wts (44/52%) and hypertrophy in ♂ and ♀ at ≥400 ppm ↑ serum alkaline phosphatase and ballooned heptocytes in ♂ and ♀ at 1600 ppm
Rat developmental toxicity, 1984, 2005 (Costlow & Kane 1984a)	0, 31.3, 93.8, 313 and 469 mkd	No developmental effects that are suggestive of an E or anti-E effect		No developmental effects that are suggestive of an E or anti-E effect		No effects	No effects	• General maternal toxicity (bloody urine, scant feces, salivation, staining of urogenital area) at ≥312.6 mkd• ↓viability index at >93.8 mkd
Rabbit developmental toxicity, 1984 (Costlow & Kane 1984b)	0, 20, 60 and 200 mkd	• ↑ abortions and resorptions at 200 mkd	No effects suggestive of an anti-E effect	No developmental effects that are suggestive of an E or anti-E effect		No effects	No effects	• ↓ BW (3%) at 200 mkd during gestation; clinical signs• Embryotoxic at 200 mkd-decreased viability index and ↓ fetal BW
Rat two-generation reproductive toxicity study, 1985 (Costlow & Harris 1985) (parental animals mated twice in each generation) – 25/sex/generation	0, 50, 200 and 1000 ppm (0, 4, 16 and 80 mkd)	• At 1000 ppm• ↓ no. ♀ delivering litters in 2 of 4 matings• Slight ↓ P2 ♀ mated (sperm +)• Slight ↑ time to mating (2 animals)• ↑ still-born pups (4 of 4 matings)		No effects	At 1000 ppm• Testicular atrophy (P2 only)• Decreased amounts of spermatozoa(P2 only)• Necrotic spermatocytes/spermatids in epididymal tubules (P2 only)• Prostate atrophy(P2 only)	Adrenals were not examined	NA	Systemic toxicity at 1000 ppm• ↓BW (9%) at 1000 ppm in P2 ♂ prior to mating (due to lower weanling wts); transient decrease in P1 ♂ from wks 1–2 pre-mating• ↓ BWG in pups during lactation at 1000 ppm (increasing effect until weaning)• Greater effects in P2 attributed to higher dosages during lactation and post-weaning periods• ↓ Litter size in 1 of 4 matings Liver: ↑ Rel wts (4–13%), hypertrophy, and vacuolization in P1 and P2 adults at ≥200 ppm (only adaptive changes in ♀)

Table 15. Triadimefon: Bioactivity concordance in mammalian toxicology assays across three lines of evidence.

Endocrine activity	HTS	Part 158 guideline studies	EDSP Tier 1 + published OSRI	Concordance
S	In vitro inhib of aromatase at concentrations that may result in cytotoxicity		In vitro inhib of aromatase	Concordant. The in vitro results of HTS and Tier 1 OSRI appear concordant, suggesting in vitro inhib of aromatase and no direct interaction with ER or AR
E Receptor binding	Negative		Negative
E Receptor transactivation	AUC model score 0.0095 (Negative)		In vitro transactivation at concentrations that may result in cytotoxicity
A Receptor binding	AUC model score 0 (Negative)		Negative
A Receptor transactivation	Negative		Negative. A published AR transactivation assay (Kojima et al. 2004) was negative for agonist and antagonist interactions
T receptor activity	Negative
In vitro anti-T activity	27/77 hepatic catabolism assays were positive for triadimefon. Assay data suggest a potential activation of hepatic nuclear receptors that could result in ↑ metabolism and clearance of T hormones in vivo. However, only two expression assays (CLZD_UGT1A1_24 and CLZD_SULT2A1_48) are linked directly to T hormone catabolism.			There are no in vitro anti-T studies available to compare to the HTS dataset HTS results showing ↑ UDPGT and SULT expression are concordant with effects observed in vivo in rat and mouse liver (including toxicogenomic data) and T effects.
In vivo E activity		↑ rel and abs ovary wt at 1800 ppm in P generation only Multigeneration study (Eiben 1984)	↑ rel ovary wt at 1800 ppm (Rockett et al. 2006).	Concordant. In vivo there are few, minor findings, including ↑ ovary wt at 1800 ppm in both guideline and Tier 1+ OSRI studies. These findings do not support the hypothesis that triadimefon exerts E-related effects; rather, these may be attributable to S effects
In vivo anti-E activity		Negative – across 11 studies in rats, mice, rabbits and dogs	Delayed VO, abnormal estrous cyclicity at 1800 ppm (Rockett et al. 2006)	In vivo, some suggestion of anti-E comes from OSRI. However, these few effects may be due to nonspecific toxicity or effects on S
In vivo A activity		↑ serum testosterone and rel testis wt in F1 at 1800 ppm (Multigeneration study, Eiben 1984)	• ↑ serum testosterone at 500 (Goetz et al. 2007) and 1800 ppm (Goetz et al. 2007, 2009)• ↑ abs/rel testes wt at mid-dose (500 ppm) (high dose data unavailable) (Goetz et al. 2007)• ↑ AGD at 1800 ppm (Goetz et al. 2007)	Concordant. ↑serum testosterone in vivo may be consistent with a perturbation of S pathways
In vivo anti-A activity		↓ F1 ♂ mating behavior at 1800 ppm Multigeneration studies (Eiben 1984, Loser & Lorke 1979)	• ↓ F1 ♂ mating behavior; delayed PPS at 1800 ppm (Goetz et al. 2007)• ↓ fecundity in FSTRA	Concordant. ↓ ♂ mating behavior in rodents at 1800 ppm was evident for both guideline and EDSP/OSRI studies
In vivo T activity		Negative – across 11 studies in rats, mice, rabbits and dogs	Negative in OSRI used in place of pubertal assays and other short-term studies	Concordant: Negative
In vivo anti-T activity		Slight ↑ T cystic hyperplasia and follicular cell adenomas at 1800 ppm (Chronic rat study, Bomhard 1991)	• ↓ serum T4 at 1800 ppm (Wolf et al. 2006; Goetz et al. 2007)• Mild ↑ follicular cell hypertrophy at 1800 ppm (Wolf et al. 2006; Rockett et al. 2006)• ↑ T colloid depletion; ↓ serum T3 and TSH at 1800 ppm, depending on exposure duration; ↑ hepatic UDPGT activity at 30 and 90 d treatment; ↑ cell proliferation in the T at 30 d (1800 ppm) (Wolf et al. 2006).	Concordant. Both dose-dependent and duration-dependent effects on the rodent T are apparent, with effects on T hormones at 1800 ppm and changes in T histopath at the same dose with long-term exposure. Findings are consistent with increased hepatic catabolism of thyroid hormones, as suggested by the in vitro anti-T HTS results

Table 16. Propiconazole: Bioactivity concordance in mammalian toxicology assays across three lines of evidence.

Endocrine activity	HTS	Part 158 guideline studies	EDSP Tier 1 + published OSRI	Concordance
In vitro Steroidogenesis (S)	In vitro inhib of aromatase at 2 uM or 24 uM		In vitro inhib of aromatase and altered S	Concordant. Both suggest inhib of S at possibly cytotoxic concentrations (>1–24 uM)
E Receptor binding	Negative		Negative. Demonstrated disruption of the binding assay at high conc. (0.6–1.2 mM)	Concordant = negative
E Receptor transactivation	AUC model scores 0 (E), 0 (anti-E) Few positive results (3/16 for E; 2/14 for anti-E) at high conc. (most ≥25 μM)		In vitro transactivation either activated (25 μM) or inhibited (52 μM) at very high concentrations	Concordant. Few positive results for interaction with ER at high concentrations (most >25 μM), likely at cytotoxic concentrations
A Receptor binding	AUC model score 0 (Negative) for A; AUC model score 0.11 (weak) for anti-A; with 5/8 assays positive in 13–70 μM range		Binding and reporter assays: generally negative for AR activation; weak positive for anti-A in vitro effects (4–96 μM range)	Concordant. Generally negative for A and weak positive in vitro for anti-A effects (4–96 μM) in vitro, likely at cytotoxic concentrations
A Receptor transactivation
T receptor activity	Negative (activation or inhib)		Not tested (not a part of EDSP Tier 1 tests)
In vitro anti-T activity	27/76 hepatic catabolism assays were positive, but most were non-specific CYP induction. Only 1 conjugation assay positive at high concentration (SULT2A1 – 48 h); no effect on UDPGT1A1 activity		Not tested (not a part of EDSP Tier 1 tests)	See in vivo assays related to T.
In vivo E activity		Negative – across 11 studies in rats, mice, rabbits and dogs	Negative – across in vivo uterotrophic, ♀ pubertal, and other studies	Concordant. Negative in the in vivo studies
In vivo anti-E activity		Negative – across 11 studies in rats, mice, rabbits and dogs	Negative in mammalian studies (♀ pubertal + OSRI)	Concordant. Negative in mammalian studies
In vivo A activity		Negative – across 11 studies in rats, mice, rabbits and dogs.	Negative in all mammalian studies, except: ↑ AGD at 2500 ppm in F1 ♂ (Goetz et al. 2007); no effect on AGD in ♀ or in several other similar studies (Taxvig et al. 2008; Rockett, 2006)	Concordant. Negative except for one non-reproducible finding
In vivo anti-A activity		Negative – across 11 studies in rats, mice, rabbits and dogs	Negative in mammalian studies (♂ pubertal, Hershberger and OSRI)	Concordant. Negative in mammalian studies
In vivo steroidogenesis		Negative – across 11 studies in rats, mice, rabbits and dogs	Negative in mammalian studies (♂ pubertal, ♀ pubertal, Hershberger + OSRI)	Concordant. Negative in mammalian studies
In vivo thyroid activity		Negative – across 11 studies in rats, mice, rabbits and dogs	Negative – in amphibian, ♂ pubertal, ♀ pubertal, Hershberger + OSRI studies	Concordant – Negative.
In vivo anti-thyroid activity		Negative – across 11 studies in rats, mice, rabbits and dogs	Negative – in amphibian, ♂ pubertal, ♀ pubertal, Hershberger + OSRI studies. One OSRI (Wolf et al. 2006): ↓ serum T4 on days 4 and 30; ↑ UDPGT activity in liver (2500 ppm), but T4, T3 and TSH normal by d 90 in rats and no T micropathology seen	Concordant – Negative One finding (lower T4 in rats at 2500 ppm) could be secondary to ↑ UDPGT activity (1-naphthol as substrate) in liver of rats; effect was transient with no functional consequence

Table 17. Myclobutanil: Bioactivity concordance across three lines of evidence.

Endocrine activity	HTS	Guideline studies	Tier 1 + OSRI package	Concordance
In vitro Steroidogenesis (S)	In vitro inhib of aromatase with one AC50 value greater than the cytotoxicity caution flag		In vitro inhib of aromatase and S	Concordant. The in vitro results of HTS, OSRI and Tier 1 appear concordant, suggesting in vitro inhib of aromatase and no direct interaction with ER or AR
E Receptor binding	Negative		Negative
E Receptor transactivation	AUC model score 0 (Negative)		Negative
A Receptor binding	AUC model score 0 (Negative)		Negative
A Receptor transactivation	Negative
T receptor activity	Negative
In vitro anti-T activity	19/79 hepatic catabolism assays were positive. Assay data suggests a potential activation of hepatic nuclear receptors that could result in ↑metabolism and clearance of T hormones in vivo			See in vivo assays related to T
In vivo E activity		• Negative ((sub-chronic and chronic studies)• ↑ rel ovary wts considered spurious and not treatment related with either no dose response or occurrence concomitant with systemic toxicity)	• Negative (across in vivo uterotrophic and ♀ Pubertal Tier 1 assays)• ↑ rel ovary wt at 2000 ppm (Rockett et al. 2006)	• Concordant. Negative. No evidence of estrogen activity exerted by myclobutanil across all the lines of evidence-Part 158, Tier 1 and or OSRI
In vivo anti-E activity		• Negative (multigen, effects seen attributed to maternal toxicity)	• Negative (Female Pubertal Tier 1 Assay) No effects on age at VO, estrous cycling, estradiol levels, ovarian or uterine wts or histopath• OSRI: ↑ AGD and delayed VO at 2000 ppm (Rockett et al. 2006)	• In vivo, some indictorss of anti-E effects from OSRI. However, these effects are likely due to nonspecific toxicity and/or effects on S. No indicators of direct anti-E potential
In vivo A activity		Negative	• Negative (Hershberger and male pubertal)• OSRI: No delay in PPS ↑ AGD, testesterone levels, ↑ testes wts and rel ventral prostate wt; reduced litter survival, impaired insemination and fertility (at 2000 ppm with toxicity) (Goetz et al. 2007)	• Concordant –Negative• Androgenic effects seen in Goetz et al. (2007) was not evident in any other studies
In vivo anti-A activity		• Effects on testes, prostate and epididymes (atrophy, ↓ amounts of spermatozoa); ↓ litter size, ↑ stillborn pups & no. ♀ deliveringat 88 mkd (2-gen Rat Study)• ↑ incidence of testicular atrophy at ≥9.84 mkd (Chronic Rat study)	• Delayed PPS (+ 1.7 d) only at 400 mkd (no change in bwt at PPS); ↓ testosterone levels and AST wts at 200 and 400 mkd (↑ abs and rel. liver wt) (male pubertal assay)• Reduced mating of F1 ♂ at 2000 ppm coincident with ♂ wt decreases (Goetz et al. 2007)	Concordant Anti-A effects were evident for part 158/EDSP and OSRI• Full database suggests altered steroidogenesis (via aromatase inhibition) accompanied by liver changes at these high-dose levels in mammals in vivo• No indications of a direct anti-A effect
In vivo T activity		Negative- across all Part 158 studies	Negative-in amphibian, pubertal assays and OSRI	Concordant.: Negative
In vivo anti-T activity		↑ rel T wt and ↑ small follicles at 3000–10000 ppm; (51.5–195 mkd) (only seen in sub-chronic rat, not seen consistently)	↑ TSH and altered histopath seen only in ♂ pubertal assay (Tier 1) only at higher dose level (400 mkd); Changes in thyroid hormones seen in only one study but no thyroid histology (Wolf et al. 2006) [↓ serum T4 only on d 4 and T3 only on d 30 (2000 ppm)]	Effects on T are not evident consistently across multiple studies. Weak effect that may be dose- and time-dependent. Effects likely secondary to ↑UDPGT hepatic enzyme induction and T hormone clearance

Table 18. AUC model and IBER values for the three triazoles.

AUC Model Scores* and IBER† values
Chemical	ER agonist	ER antagonist	ER IBER	AR agonist	AR antagonist	AR IBER
Myclobutanil	0	0	1,000,000	0	0	1,000,000
Propiconazole	0	0	1,000,000	0	0.111	643,056
Triadimefon	0.0095 (negative; belowthreshold)	0	1,000,000	0	0	1,000,000

* AUC model, area-under-the-curve model for ER or AR pathway activity.

† IBER, integrated bioactivity and exposure ranking. From Browne, et al. (2015) and USEPA (2014b).

Table 19. AC₅₀ values in HTS aromatase inhibition assays for the three triazoles

	Triadimefon		Propiconazole		Myclobutanil
Assay endpoint name	μM	OED*	μM	OED	μM	OED
NVS_ADME_hCYP19A1	2.06	0.67	2.24	2.5	0.67	0.38
Tox21_Aromatase_Inhibition	36.8	12	23.8	26	5.16	2.9

* OED, oral equivalent dose, mkd, calculating using the Css95% (Table 1).

Table 20. AC₅₀ values in HTS assays for UGT1A1 and SULT2A1 induction.

	Triadimefon		Propiconazole		Myclobutanil
Assay endpoint name	μM	OED*	μM	OED	μM	OED
CLZD_SULT2A1_48	8.29	2.7	7.44	8.2	NA	NA
CLZD_UGT1A1_24	9.66	3.2	NA†	NA	14.6	8.3

* OED, oral equivalent dose, mkd, calculating using the Css95% (Table 1).

† NA, tested but negative in this assay.

Figure 5. Proposed roadmap for EDSP evaluation of pesticide active ingredients (PAIs). For PAIs, the available high-throughput (HT) hazard and exposure information would be considered first, followed by evaluation of the available guideline toxicology study information before making any priority decisions. Priority decisions could include decisions to collect additional hazard or exposure information, or to make a risk assessment (RA) decision based on adequate available data or suitable margins of separation between predicted exposure and bioactivity.

USEPA. 2014b. Integrated Bioactivity and Exposure Ranking: A Computational Approach for the Prioritization and Screening of Chemicals in the Endocrine Disruptor Screening Program. Available from: http://www.regulations.gov/#!documentDetail;D = EPA-HQ-OPP-2014-0614-0003.

 Google Scholar

Wambaugh JF, Wang A, Dionisio KL, Frame A, Egeghy P, Judson R, Setzer RW. 2014. High throughput heuristics for prioritizing human exposure to environmental chemicals. Environ Sci Technol. 48:12760–12767.

 PubMed Web of Science ®Google Scholar

USEPA. 2008. Mycobutanil: Amended Human Health Risk Assessment for Proposed Use on Section 3 Requests for Use on Snap Bean, Mint, Papaya, Gooseberry, Currant, Caneberry, Bell and Non-Bell Pepper, Head and Leaf Lettuce, and Artichoke PC Code 128857 DP Barcode: 341689.

 Google Scholar

USEPA. 2014d. Propiconazole Human Health Risk Assessment for the Section 3 Registration on Rapeseed Crop Subgroup 20A.

 Google Scholar

USEPA. 2009. Triadimefon: Human Health Risk Assessment to Support Reinstatement of Use on Residential Turf Decision No. 391164.38.

 Google Scholar

Goetz AK, Ren H, Schmid JE, Blystone CR, Thillainadarajah I, Best DS, Nichols HP, Strader LF, Wolf DC, Narotsky MG, et al. 2007. Disruption of testosterone homeostasis as a mode of action for the reproductive toxicity of triazole fungicides in the male rat. Toxicol Sci. 95:227–239.

 PubMed Web of Science ®Google Scholar

Wolf DC, Allen JW, George MH, Hester SD, Sun G, Moore T, Thai SF, Delker D, Winkfield E, Leavitt S, et al. 2006. Toxicity profiles in rats treated with tumorigenic and nontumorigenic triazole conazole fungicides: propiconazole, triadimefon, and myclobutanil. Toxicol Pathol. 34:895–902.

 PubMed Web of Science ®Google Scholar

Rockett JC, Narotsky MG, Thompson KE, Thillainadarajah I, Blystone CR, Goetz AK, Ren H, Best DS, Murrell RN, Nichols HP, et al. 2006. Effect of conazole fungicides on reproductive development in the female rat. Reprod Toxicol. 22:647–658.

 PubMed Web of Science ®Google Scholar

Bauer ER, Bitsch N, Brunn H, Sauerwein H, Meyer HH. 2002. Development of an immuno-immobilized androgen receptor assay (IRA) and its application for the characterization of the receptor binding affinity of different pesticides. Chemosphere. 46:1107–1115.

 PubMed Web of Science ®Google Scholar

Kojima H, Katsura E, Takeuchi S, Niiyama K, Kobayashi K. 2004. Screening for estrogen and androgen receptor activities in 200 pesticides by in vitro reporter gene assays using Chinese hamster ovary cells. Environ Health Perspect. 112:524–531.

 PubMed Web of Science ®Google Scholar

Ohno K, Araki N, Yanase T, Nawata H, Iida M. 2004. A novel nonradioactive method for measuring aromatase activity using a human ovarian granulosa-like tumor cell line and an estrone ELISA. Toxicological Sci. 82:443–450.

 PubMed Web of Science ®Google Scholar

USEPA. 2007b. Myclobutanil: Acute and Chronic Food and Drinking Water Dietary Exposure and Risk Assessments for Section 3 Use on Snap Bean, Mint, Papaya, Gooseberry, Currant, Caneberry, Bell and Non-Bell Pepper, Head and Leaf Lettuce, and Artichoke PC Code 128857 DP 341690.

 Google Scholar

Kjaerstad MB, Andersen HR, Taxvig C, Hass U, Axelstad M, Metzdorff S, Vinggaard AM. 2007. Effects of azole fungicides on the function of sex and thyroid hormones. Pesticides Research No. 111.

 Google Scholar

Hurst MR, Sheehan DA. 2003. The potential for oestrogenic effects of pesticides in headwater streams in the UK. Sci Total Environ 301:87–96.

 PubMed Web of Science ®Google Scholar

Taxvig C, Vinggaard AM, Hass U, Axelstad M, Metzdorff S, Nellemann C. 2008. Endocrine-disrupting properties in vivo of widely used azole fungicides. Int J Androl. 31:170–177.

 PubMed Web of Science ®Google Scholar

Tully DB, Bao W, Goetz AK, Blystone CR, Ren H, Schmid JE, Strader LF, Wood CR, Best DS, Narotsky MG, et al. 2006. Gene expression profiling in liver and testis of rats to characterize the toxicity of triazole fungicides. Toxicol Appl Pharmacol. 215:260–273.

 PubMed Web of Science ®Google Scholar

Goetz AK, Rockett JC, Ren H, Thillainadarajah I, Dix DJ. 2009. Inhibition of rat and human steroidogenesis by triazole antifungals. Syst Biol Reprod Med. 55:214–226.

 PubMed Web of Science ®Google Scholar

Willoughby JA. 2012b. Triadimefon: estrogen receptor binding (rat uterine cytosol)-amended report 1. Kalamazoo (MI): Unpublished study prepared by CeeTox, Inc. MRID 48619602:81.

 Google Scholar

Willoughby JA. 2012a. Triadimefon: androgen receptor binding (rat prostate cytosol)-amended report 1. Kalamazoo (MI): Unpublished study prepared by CeeTox, Inc. MRID 48619602:81.

 Google Scholar

Willoughby JA. 2012c. Triadimefon: estrogen receptor transcriptional activation (human cell line HeLa-9903)-amended report 1. Kalamazoo (MI): Unpublished study prepared by CeeTox, Inc. MRID 48619605:63.

 Google Scholar

USEPA. 2007a. Integrated Summary Report on Aromatase.

 Google Scholar

Trosken ER, Scholz K, Lutz RW, Volkel W, Zarn JA, Lutz WK. 2004. Comparative assessment of the inhibition of recombinant human CYP19 (aromatase) by azoles used in agriculture and as drugs for humans. Endocr Res. 30:387–394.

 PubMed Web of Science ®Google Scholar

Davis J. 2011. The Hershberger bioassay (OPPTS 890.1400); triadimefon. Unpublished study prepared by ILS, Integrated Laboratory Systems, Inc. MRID 48619604:107.

 Google Scholar

Laville NP, Brion F, Hinfray N, Casellas C, Porcher J-M, Ait-Aissa S. 2006. Modulation of aromatase activity and mRNA by various selected pesticides in the human choriocarcinoma JEG-3 cell line. Toxicology. 228:98–108.

 PubMed Web of Science ®Google Scholar

Trosken ER, Fischer K, Volkel W, Lutz WK. 2006b. Inhibition of human CYP19 by azoles used as antifungal agents and aromatase inhibitors, using a new LC-MS/MS method for the analysis of estradiol product formation. Toxicology 219:33–40.

 PubMed Web of Science ®Google Scholar

Sanderson, IT, Boerma J, Lansbergen GWA, van den Berg M. 2002. Induction and Inhibition of aromatase (CYP 19) activity by various classes of pesticides in H295R human adrenocortical carcinoma cells. Toxicol Appl Pharmacol. 182, 44–54.

 PubMed Web of Science ®Google Scholar

Coder P. 2012. Propiconazole – uterotrophic assay in ovariectomized rats (OECD guideline 440; US EPA OPPTS 890.1600): Unpublished study prepared by WIL Research Laboratories, LLC, Ashland, OH USA. MRID 48619003.

 Google Scholar

LeBaron MJ, Schisler MR, Visconti NR. 2011a. Evaluation of myclobutanil in an in vitro estrogen receptor binding assay. Unpublished study prepared by Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, MI. Laboratory Study No.: 111118, December 14, 2011. MRID 48618104.

 Google Scholar

LeBaron MJ, Schisler MR, Visconti NR. 2011b. Evaluation of myclobutanil in an in vitro androgen receptor binding assay. Unpublished study prepared by Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, MI. Laboratory Study No.: 111113, December 5, 2011. MRID 48618102.

 Google Scholar

LeBaron MJ, Kan HL. 2011c. Evaluation of myclobutanil in an in vitro estrogen receptor transcriptional activation assay in human cell line hERα-HeLa-9903. Unpublished study prepared by Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, MI. Laboratory Report No.: 101193, December 12, 2011. MRID 48618105.

 Google Scholar

Coady KK, Sosinski LK. 2011. Myclobutanil: evaluation of myclobutanil in the human recombinant aromatase assay. Unpublished study prepared by Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, MI. Laboratory Project Study ID No.: 111015, November 28, 2011. MRID 48618103.

 Google Scholar

LeBaron MJ, Kan HL, Perala AW. 2011d. Evaluation of myclobutanil in the in vitro steroidogenesis assay. Unpublished study prepared by Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, MI. Laboratory Study No.: 101192, December 5, 2011. MRID 48618110.

 Google Scholar

Marty MS, Brooks KJ. 2011. Myclobutanil: uterotrophic assay in the immature female Crl:CD(SD) rat. Midland (MI): Unpublished study prepared by Toxicology & Environmental Research Consulting of The Dow Chemical Company. MRID 48618111.

 Google Scholar

Marty MS, Andrus AK, Stebbins KE. 2011. Myclobutanil: pubertal development and thyroid function in intact juvenile/peripubertal male Crl:CD(SD) rats. Midland (MI): Unpublished study prepared by Toxicology & Environmental Research Consulting of The Dow Chemical Company. MRID 48618109.

 Google Scholar

Marty MS, Marshall VA, Jeong YC, Sura R. 2011. Myclobutanil: Hershberger Assay in Castrated Male Crl:CD(SD) Rats. Midland (MI): Unpublished study prepared by Toxicology & Environmental Research Consulting of The Dow Chemical Company. MRID 48618107.

 Google Scholar

Mohr U. 1976. Meb 6447: Subchronic Toxicity Study on Rats (Twelve-week Feeding Experiment): Report No. R 840a; Report No. 44300. Unpublished study received Aug 15, 1977 under 3125-318; prepared by Medizinische Hochschule Hannover, Abteilung fur Experimentalle Pathologie, West Germany, submitted by Mobay Chemical Corp, Kansas City, Mo; CDL: 2311312-C.

 Google Scholar

Sheets LP, Gilmore R, Hoss H. 2008. A developmental neurotoxicity study with technical grade triadimefon in wistar rats. Project Number 07/D72/IL, 201766. Unpublished study prepared by Bayer CropScience. p. 1179.

 Google Scholar

Bomhard E, Schilde B. 1991. MEB 6447: Chronic toxicity and cancerogenicity studies on Wistar rats with administration in diet over a period of 105 weeks: lab project number: 20774: 101922. Unpublished study prepared by Bayer AG, Dept of Toxicology. MRID 42153901:1029 p.

 Google Scholar

Hoffman K, Groning P. 1978. Meb 6447 long-term toxicity study on dogs (two-year feeding study): Report No. 7882; Report No. 66656. Unpublished study received Apr 28, 1980 under 3125-318; prepared by Bayer, AG, submitted by Mobay Chemical Corp, Kansas City: Mo; CDL: 099413-D. MRID 32539.

 Google Scholar

Nagumo K, Teraki Y, Chiba T. 1981. Teratogenicity test of MEB 6447 in pregnant rats: submitter 80257. Unpublished study received Jan 5, 1982 under 1F2474; prepared by St Marianna Univ, First Dept of Anatomy, Laboratory of Embryology, submitted by Mobay Chemical Corp, Kansas City, Mo; CDL:070570-A. MRID 89023.

 Google Scholar

Unger T, Van Goethem D, Shellenberger T. 1982. A teratological evaluation of bayleton in mated female rats: MRI Project No. 7272-B. Unpublished study prepared by Midwest Research Institute.MRID 149336:52 p.

 Google Scholar

Clemens G, Jasty V, Troup C, Hartnagel R. 1990. A toxicity study in the rabbit with MEB 6447 (Triadimefon): Report No. 100035. Upublished study prepared by Miles Laboratories, Inc. MRID 41616005: 179 p.

 Google Scholar

Loser E, Lorke D. 1979. Meb 6447 Multigeneration Reproduction Study on Rats: Report No. 8297; Report No. 67752. Unpublished study received Apr 28, 1980 under 3125-318; prepared by Bayer, AG, submitted by Mobay Chemical Corp, Kansas City: Mo; CDL: 099413-F. MRID 32541.

 Google Scholar

Eiben R. 1984. MED 6447 (Triadimefon): Two-Generation Study with Rats: Supplementary Study: Report No. 12712. Unpublished study prepared by Bayer AG, Institute of Toxicology. MRID 155075:168p.

 Google Scholar

Sachsse K, Suter P, Luetkemeier H. 1979a. CGA-64250 Techn. Three months toxicity study on rats: Project No. 790014. Final rept. Unpublished study prepared by Ciba Geigy Ltd., Switzerland, Syngenta File No. 64250/1538. MRID 00058606.

 Google Scholar

Sachsse K, Bathe R, Luetkemeier H. 1979b. CGA-64250 3-month toxicity study on dogs: Report No. 78/5751. CDL: 244271-R. Final rept. Unpublished study prepared by Ciba Geigy Ltd., Switzerland. Syngenta File No. CGA64250/1539. MRID 00058607.

 Google Scholar

Hunter B, Slater N, Heywood R. 1982a. CGA 64 250: potential tumorigenic and toxic effects in prolonged dietary administration to rats: CBG 193/8284 (Test No. 789023). Final rept. Unpublished study prepared by Huntingdon Research Centre, England. MRID 00129918.

 Google Scholar

Hunter B, Scholey D, Haywood R. 1982b. CGA 64 250: long-term feeding study in mice. CBGI195/81827. Final Report. Unpublished study prepared by Huntingdon Research Centre, England. MRID 00129570.

 Google Scholar

Johnson W, Thompson S. 1985. One-year subchronic oral toxicity study in Beagle dogs with CGA-64250 technical (Final Report). FDRL Study No. 7737. Unpublished study prepared by Research Laboratories, Inc. 570 p. Syngenta File No. CGA64250/1544. MRID 00151515.

 Google Scholar

Fritz H. 1984a. Expanded report on CGA-64250 technical segment II teratology study in rats. Project No. 790011, 21 September 1984. Unpublished study prepared by Ciba-Geigy. Syngenta File No. CGA64250/2150. MRID 00058604.

 Google Scholar

Giknis M. 1987. CGA 64250 technical: teratology (segment II) study in rats. Project No.86004. Syngenta File No. CGA64250/1586. Unpublished study prepared by Ciba-Geigy. MRID 40425001.

 Google Scholar

Mallow S. et al. 1987. CGA 64250: a modified teratology (segment II) study in albino rats. Project No.86189. Syngenta File No. CGA64250/1587. Unpublished study prepared by Ciba-Geigy. MRID 40425002.

 Google Scholar

Fritz H. 1984b. Expanded report on CGA64250 technical segment II reproductive study in rabbits. Project No. 790009. Unpublished study prepared by Ciba-Geigy. Syngenta File No. CGA64250/2151. MRID 00058605.

 Google Scholar

Raab D, Youreneff M, Giknis M, et al. 1987. Propiconazole: a teratology study in New Zealand rabbits: final report amendment No. 1: Laboratory Project ID 86043. Unpublished study prepared by Ciba-Geigy. Syngenta File No. CGA64250/1589. MRID 40425004.

 Google Scholar

Borders C, Salamon C. 1985. Two-generation reproduction study in albino rats with CGA-64250 technical. Unpublished study prepared by Toxigenics, Inc. MRID 00151514.

 Google Scholar

Goldman P, Harris J, Lampe K. 1986a. RH-3866: a three-month dietary toxicity study in mice: Report No. 83R-136. Unpublished study prepared by Rohm & Haas Co. 522 p. MRID 00165245.

 Google Scholar

O’Hara G, DiDonato L. 1984. RH-3866: three month dietary toxicity study in rats: Final Report: Report No. 83R-068. Unpublished study prepared by Rohm and Haas Co. 702 p. MRID 00145048.

 Google Scholar

Shimizu Y. 1987. A thirteen week feeding subchronic study of RH-3866 in rats. Report No. NRILS 86-2024;R & H Report No 87RC-038. Unpublished study from NRI Life Science, Kanagawa, Japan and prepared by Rohm & Haas Co. 09 July 1987.

 Google Scholar

McLaughlin JE, DiDonato LJ 1984. RH-3866: a three month dietary study in dogs. Unpublished study report prepared by Rohm and Hass Company, a wholly-owned subsidiary of The Dow Chemical Company, Midland, Michigan.

 Google Scholar

Shellenberger T, Billups L. 1986. Chronic toxicity and oncogenicity study with RH 3866 in rats: final report: Project No. 8342. Unpublished study prepared by Rohm & Haas Co. Project No. 85RC-61 prepared by Tegeris Laboratories, Inc. 3536 p. MRID 00165247.

 Google Scholar

Wolfe GW. 1993. RH-3866 technical: 104-week dietary oncogenicity toxicity study in rats. Report No. 89RC-260Unpublished study prepared by Rohm & Haas Co. 522 p. MRID 42809101.

 Google Scholar

Goldman P, Harris J. 1986. RH-3866: Dietary chronic and oncogenicity study in mice: Final Report: Report Number 84R-023. Unpublished study prepared by Rohm and Haas Co. 3326 p. MRID 00164990.

 Google Scholar

Andersen DM, O’Hara, GP, Brown WR. 1993. RH-3866: dietary oncogenicity study in female mice. final report: Report No. 89R-261. Unpublished study prepared by Rohm and Haas Co. MRID 42809102.

 Google Scholar

Goldman P, Harris J, Frantz J. 1986b. RH-3866: one year dietary study in Beagle dogs: Protocol No. 84P-203: Report No. 84R-078. Unpublished study prepared by Rohm & Haas Co. 775 p. MRID 00165248.

 Google Scholar

Costlow R, Kane W. 1984a. Teratology study with RH-53,866 in rats: Final Report: Report No. 83R-024. Unpublished study prepared by Rohm and Haas Co. 123 p. MRID 00141672.

 Google Scholar

Costlow R, Kane W. 1984b. Teratology study with RH-53, 866 in rabbits: Report No. 83R-217. Unpublished study prepared by Rohm and Haas Co. 139 p. MRID 00164971.

 Google Scholar

Costlow R, Harris J. 1985. RH-53,866: two generation reproduction study in rats: Report No. 84R-117. Unpublished study prepared by Rohm and Haas Co. 225 p. MRID 00149581.

 Google Scholar

Browne P, Judson RS, Casey W, Kleinstreuer N, Thomas RS. 2015. Screening chemicals for estrogen receptor bioactivity using a computational model. Environ Sci Technol.

 Web of Science ®Google Scholar