# US EPA Science Advisory Panel Meeting

Comments on “ Male and Female Pubertal Assays Used in EDSP Tier 1 Screening”

Sue Marty, Ph.D., D.A.B.T. Senior Toxicology Leader The Dow Chemical Company mmarty@dow.com May 22, 2013

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## Male and Female Pubertal Assays

• Charge Question #1: Lab proficiency: How well were assays executed relative to the test guidelines and performance criteria?



### • Justification to include performance criteria was sound

• Ability to meet defined CVs is determined by variance in assay performance (proficiency) and inherent biological variability (Elswick et al., 2000)

• Based on Tier 1 data, some performance criteria may need adjusted.

• Female pubertal assay:

– Terminal body weights had CVs above the acceptable range

– Adrenal weight lower than recommended mean (11 of 16 chemicals; 69%)

– 16 of 17 labs (94%) had ≥1 parameter outside performance criteria

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## Male and Female Pubertal Assays

• Charge Question #1: Lab proficiency: How well were assays executed relative to the test guidelines and performance criteria?

– Based on available data, some performance criteria may need adjusted:

• Male pubertal assay: 32 performance criteria examined across 15 data sets

• All male assays had ≥2 parameters outside the performance criteria

Parameter CV outside
Performance Criteria Parameter Mean outside
Performance Criteria

Seminal vesicle wt.* 8 (53%) Thyroid wt. + 7 (47%)
Ventral prostate wt.* 4 (27%) Kidney wt. + 12 (80%)

Serum TSH 7 (47%) Serum TSH 5 (33%)
Pituitary wt.* 5 (33%)

Initial body wt. + 5 (33%)

Final body wt.* 12 (80%)

Body wt at PPS* 12 (80%)
Age at PPS* 5 (33%)

* Missed in 2

- 4 of 4 labs during interlaboratory validation; + Missed in 1 of 4 labs during interlaboratory validation

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## Male and Female Pubertal Assays

• Charge Question #1: Lab proficiency: How well were assays executed relative to the test guidelines and performance criteria?



### • Accuracy of performance criteria reported?

– Errors in CV reporting in both male and female pubertal assay section

– Omissions for some compounds that missed CV performance criteria



### • EPA: Consider re

- examining the performance criteria with List 1 data sets

• “In general, however, missing one or two performance criteria will not be regarded as fatal to the study.”

• Agency should provide additional guidance on how performance criteria will
be used.

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## Male and Female Pubertal Assays

• Charge Question #8: Minimum number of screens and tests to determine whether the compound has the potential to interact with the endocrine system (money, resources, time).



### – Neither the male nor female pubertal assay are rapid or cost effective

• Rats are born in house (assays ~ 53 or 63 days in length with 20

- to 30

- days of dosing and data collection for each assay)

• Fewer animals are used to collect data than are euthanized with no data collection (120 pups and 15 dams discarded vs. 90 weanlings used for data collection)

• Reporting time is ~6 months

• Cost is $85,000

- 100,000 per assay

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## Other Observations on the Pubertal Assay Evaluation in SAP Report

• Hormone measurements

– Changes in hormone measurements in the absence of any corresponding changes in other assay endpoints?

– Hormone measurements are highly variable, represent a single point in time and can be subject to rapid stress

- related changes.

– If hormone changes are sustained, why are there no additional changes
over the 20

- to 30

- day dosing periods in these highly sensitive assays?



### • Statistical analyses

– Multiple statistical analyses on the same data? (e.g., organ weights require an ANOVA, ANCOVA and linear trend test)

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## Other Observations on the Pubertal Assay Evaluation in SAP Report

• Male and female pubertal assay require some endpoints whose utility is unclear . Discussion is warranted on the value of:

– Ovarian and uterine weights in cycling female rats

– Both wet and blotted uterine weights and seminal vesicle weights

– Five point grading scale for thyroid histopathology

– Estrous cycle data (highly variable and limited monitoring period)



### • Diet affects female pubertal assay results:

– Dietary phytoestrogens below 350 μg/g genistein equivalents

– Most labs to use low phytoestrogen

- containing diets. This has

– Altered the age at vaginal opening in many labs (approximately +2 days vs. two

- generation studies).

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## Male and Female Pubertal Assays

• EDSP Tier 1 Battery Performance: Evaluate “complementarity” relationships or concordance among endpoints within an assay

• For the Male and Female pubertal assays:

Pubertal assays detect MoAs not detected in other Tier 1 assays and species/gender differences in response may be meaningful; therefore, will all of these compounds progress to Tier 2? Assay N Positive Results Systemic Tox /
Solvent Interference Positive without Systemic Toxicity

Female Pubertal 17 12 (71%) 4 8 (47%)

Male Pubertal 16 10 (63%) 2 8 (50%)

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## Male and Female Pubertal Assays

• EDSP Tier 1 Battery Performance: Evaluate “redundancy” or concordance among endpoints across assays?

• In discussions on the androgen pathway (pp. 141

- 146), it states that the aromatase assay provides information on androgen pathway interactions. While conceptually possible, the in vivo mammalian assays identified as sensitive to androgens are not good at detecting aromatase inhibitors.

– Hershberger assay detects aromatase activity with mixed modes of action (e.g., antiandrogenicity as reported with fenarimol)

– Specific aromatase inhibitor, fadrozole, was not detected in the male pubertal

assay

– EPA concluded, “...the results with fadrozole suggest that compounds specific to aromatase inhibition will not be detected with this assay”.

– Please clarify how effects on aromatase will contribute to understanding effects on androgen signaling

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## Male and Female Pubertal Assays

• EDSP Tier 1 Battery Performance: Evaluate “redundancy” or concordance among endpoints across assays?

• When combining all assays:

Pathway N Positive Results Systemic Tox /
Solvent Interference Positive without Systemic Toxicity
Thyroid 16 13 (81%) 5 8 (50%)*

Androgen 17 17 (100%) 3 14 (82%)

Estrogen 18 18 (100%) 8 10 (56%)

*Three detected in only one assay; some due to hormone changes alone.

• Endocrine

- sensitive endpoints were altered across assays, but there is insufficient data to determine whether changes fit a pattern of responses (e.g., were results in the female pubertal assay and FSTRA consistent for a similar MoA).

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## Male and Female Pubertal Assays

• EPA has concluded that the battery has performed as anticipated. Please
comment on this conclusion.

• The male and female pubertal assays are highly sensitive to detect potential endocrine activity as illustrated by the high rate of positives in these assays (47

- 50%). Reliance on apical endpoints may make it difficult to determine MoA for endocrine

- related findings.

• The pubertal assay detects compounds that interact at ER/AR, affect steroidogenesis , alter HPG or HPT axis function, induce hepatic enzymes and
enhance thyroxine or steroid hormone clearance, as well as non

- specific results due to systemic toxicity (e.g., 6% decrease body weight gain), stress, etc. Variability in some endpoints (e.g., hormone measurements, estrous cyclicity , etc.) can complicate interpretation.

• The specificity and toxicological relevance of these assays remains questionable.

• If gender and species differences are meaningful, it is difficult to dismiss a Tier 1 pubertal assay positive, because these assays detect different MoAs not detected by other Tier 1 assays.

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