**[October 9 - 12, 2007 FIFRA scientific advisory panel]{.smallcaps}**

**[The potential for atrazine to affect amphibian gonadal
development]{.smallcaps}**

**[Charge to the Panel]{.smallcaps}**

The Agency has reviewed relevant studies in the scientific literature
published since 2003, as well as studies submitted by the registrant in
response to a DCI, to evaluate the potential for atrazine alone to
elicit effects on amphibian gonadal development and differentiation. The
strengths and limitations of the individual studies were assessed, and
the extent of concordance for the entire body of information derived
from these laboratory and field studies was considered to assess the
plausibility that atrazine can cause developmental effects in amphibian
gonads, and if so, the nature and strength of associated dose-response
relationships. This analysis, while primarily focused on gonadal
development and differentiation, included data on other developmental
effects, including: time to metamorphosis, growth, sex ratios, and
gonadal abnormalities.

The Agency is seeking comments and recommendations from the SAP on a
number of questions surrounding the current body of evidence regarding
the potential effects of atrazine on gonadal development in amphibians
and the relevancy of these potential effects to an ecological risk
assessment of atrazine. The Agency is also seeking input from the SAP on
its conclusion that, based on the results of the most recent studies in
response to the DCI, atrazine exposure does not affect amphibian gonadal
development and that no further testing is required to address this
hypothesis.

**[Questions]{.underline}**

1)  In reviewing the available laboratory and field studies, the Agency
    used a number of criteria to evaluate individual investigations.
    Criteria such as experimental design, test protocols, and quality
    assurance information were used to evaluate the reliability of a
    study's ability to adequately assess a hypothesis that atrazine
    elicits developmental effects in amphibians, and if so, the nature
    and strength of associated dose-response relationships.

> **(a)** Please provide comments and recommendations regarding the
> EPA's approach and criteria used to evaluate the studies.

**(b)** Given the evaluation criteria employed by the Agency, please
comment on EPA's overall application of these criteria to the currently
available studies.

**[Questions Concerning the Open Literature Studies]{.underline}**

**2)** The Agency has concluded that the information contained in the
open literature published since the 2003 SAP does not provide any
additional information that could be used to refute or confirm the
hypothesis that exposure to atrazine alone causes adverse developmental
effects in amphibian gonads.

> **(a)** Please comment on the comprehensiveness of the Agency's
> literature reviews relative to the potential effects of atrazine alone
> on amphibian gonadal development.
>
> **(b)** Please comment on the Agency's evaluation of the open
> literature studies and the Agency's conclusion that the data derived
> from laboratory studies, both individually and collectively, are not
> sufficient to refute or confirm the hypothesis that atrazine exposure
> causes developmental effects in amphibian gonads.
>
> **(c)** The Agency concluded that the field studies are not adequate
> for assessing the hypothesis at hand. Please comment on the Agency's
> conclusion. If the SAP concludes one or more of the field studies do
> provide the means to assess the hypothesis that atrazine exposure
> results in effects on amphibian gonadal development, please suggest
> interpretive and statistical methods that should be employed to
> evaluate the data.

**[Questions Concerning the DCI Study]{.underline}**

**3)** Please comment on the Agency's evaluation of the final study
design. For example, the Agency concluded that the minor changes in the
experimental design \[*i.e*., omitting atrazine degradate (DACT, DEA and
DIA) analysis and not conducting differential cell counts for ovarian
and testicular histology\] did not compromise the means to assess the
hypothesis that atrazine exposure can affect amphibian gonadal
development. If the SAP concludes that the alterations in the study
design preclude or significantly compromise the ability to assess the
hypothesis, please discuss to the extent possible, how the specific
design modifications could impact the means to assess the hypothesis.
Please provide comments on other aspects of the Agency's evaluation as
well.

**4)** The Agency has described the exposure profiles for studies
conducted in response to the DCI and has stated that mean-measured
concentrations in the studies were lower than target nominal
concentrations. However, the Agency concluded that the frequent
analytical measurements provide a sufficiently comprehensive
understanding of the exposure profile over the course of the studies.
Please comment on the Agency's conclusion that the atrazine exposure
concentration profile is reasonably characterized and sufficient for
documenting the potential effects of atrazine over a broad range of
exposure concentrations. In addition, provide comments on whether the
actual concentrations were consistent and sufficiently stable to
establish the means to analyze exposure concentration-response
relationships.

**5)** The Agency described atrazine contamination of negative controls
in one out of the two studies and concluded that since the experimental
design had twice the number of controls relative to other treatments,
the data from these atrazine-contaminated controls could be removed from
the analyses without invalidating the statistical interpretation of the
results.

**(a)** Please comment on the Agency's decision to omit half of the
controls from the WLI study in the statistical analyses and on the
conclusion that the study is still scientific valid. If the SAP has an
alternative approach to treating these control data in the statistical
analyses, please provide specific recommendations.

**6)** The original White Paper (USEPA 2003) identified measurement
endpoints that included the possible enumeration of specific
histological structures such as the number of oogonia in ovaries and the
number of spermatids in testes. Such a detailed analysis was not
conducted in the studies that are in response to the DCI. Rather, a
qualitative assessment of the incidence of ovarian and testicular
gonadocytes was conducted. The Agency has concluded that the lack of
these data does not limit the means to assess the hypothesis that
atrazine exposure affects amphibian gonadal development.

**(a)** Please comment on the whether the lack of these histological
data limits the utility of the available information to fail to support
the hypothesis that atrazine exposure affects amphibian gonadal
development.

(**b)** If the SAP concludes these data are necessary to adequately
assess the hypothesis, please provide options to processing and
analyzing these data in an efficient and robust manner.

> **7)** The Agency has described a number of measurement endpoints
> (*e.g*., translucent gonads, unpigmented ovaries, pigmented testes)
> based on histology results that were reported in the studies. The
> Agency, however, based on its understanding of relevant scientific
> literature, could not conclude that these measurement endpoints are
> biologically relevant indicators of effects on growth or reproductive
> success (*i.e.,* the Agency did not interpret these responses as
> adverse effects *per se*) nor was the Agency aware of any information
> that established these responses as precursors to the apical endpoints
> of primary interest (*i..e*., time to and size at metamorphosis, sex
> ratio, and the presence of mixed and/or intersex animals).
>
> **(a)** Please comment on the biological relevancy of these endpoints
> and the extent to which they may reflect reliable measures of
> developmental abnormalities.

**8)** The Agency's analysis of potential developmental effects in
studies responsive to the DCI has focused on histological data as
opposed to gross morphological data. The histological data from these
studies are based on the analyses of a single certified pathologist.
While this approach eliminates the potential variability associated with
having multiple pathologists analyze the histological slides, it may
introduce an avidity bias.

(**a)** Please comment on whether a single pathologist is sufficient for
interpreting the histology data. If the SAP believes that a single
pathologist is not sufficient, please comment on the potential value of
convening a pathology review board to evaluate the findings of the DCI
study.

**(b)** Please also comment on the potential value of having a pathology
review board evaluate materials (*e.g*., digital images of gross
morphology and microscope slides containing histological sections) from
studies published in the open literature. These data could be submitted
voluntarily by the authors and could include slides to evaluate
similarities or differences in identifying or describing histological
features and/or describing and quantifying histological responses.

**9)** After an evaluation of the laboratory-based studies submitted in
response to the DCI, the Agency has concluded that these studies do not
provide sufficient evidence to support the hypothesis that atrazine
causes adverse gonadal developmental effects in amphibians.

**(a)** In light of the responses to Questions 3 -- 8, please comment on
whether the results from the study in response to the DCI are
sufficiently robust to address the hypothesis that atrazine exposure
causes gonadal abnormalities in *X. laevis*. If the SAP concludes these
results are not sufficiently robust, what recommendations can the SAP
provide to efficiently and reasonably address remaining uncertainties?
For example, if the SAP does not believe the DCI study is sufficiently
robust to assess the hypothesis, does the SAP believe either the two
experiments or a specific component of the two experiments should be
reanalyzed or repeated? Please provide the rationale for recommending
any additional analyses and/or experiments.

**(b)** Please comment and provide recommendations on alternate
statistical analyses, if any, to evaluate the data derived from the
study. If alternative approaches are suggested, please comment, to the
extent possible, on the rationale for these approaches and how they
represent improvements in the existing statistical interpretations.

**[Concluding Questions]{.underline}**

**10)** Is the SAP aware of any other laboratory-based or field-based
studies not included in this White Paper that contradict the Agency's
conclusions that 1) the designs associated with current studies
available in the open literature are not appropriate for evaluating the
hypothesis that atrazine affects amphibian gonadal development and 2)
the available data in the open literature combined with the results of
DCI study indicate that atrazine does not cause adverse effects on
gonadal development in *X. laevis* when investigated under conditions
consistent with those recommended by the SAP in its previous report (SAP
2003). If so, please identify the studies and briefly outline how the
results from these studies would contradict the conclusion that atrazine
at concentrations up to 100 μg/L does not cause adverse effects on
amphibian gonadal development.

**11)** The Agency is not aware of data that establish a mechanistic
basis for how atrazine could affect amphibian gonadal development.
Please identify and comment on any studies that demonstrate the
mechanistic steps by which amphibian gonadal development could be
affected by atrazine, and thereby contradict the Agency's overall
conclusions based on the studies evaluated for this SAP review. If the
SAP is aware of any relevant study(ies), please comment on the data from
this study(ies) and how the data indicate and quantify a mechanistic
pathway from atrazine's molecular site of action to histological and
apical endpoints associated with adverse effects on amphibian gonadal
development. Please also comment on any dose-response relationships
associated with the steps in the reported toxicity pathway.

**12)** In its 2003 White Paper the Agency proposed a research approach
using focused, empirical laboratory studies based on initial
investigations with *X. laevis,* potentially followed by selective,
confirmatory laboratory studies with frog species native to North
America. However, the 2003 SAP did not identify any important
differences between amphibian species to conclude that any affected
developmental and/or mechanistic processes observed in *X. laevis* would
not be applicable to indigenous ranid species.

**(a)** Please comment on the Agency's recommendation that data derived
from *X. laevis* in the studies evaluated for this review are sufficient
to conclude that additional testing with indigenous species is not
warranted.

13) Based on the available data provided by the DCI studies, the Agency
    has concluded that atrazine does not adversely affect amphibian
    gonadal development. The Agency has further concluded that no
    additional studies are required to address the hypothesis that
    atrazine adversely affects amphibian gonadal development.

    a.  Please comment on the Agency's recommendation that the current
        body of data is sufficient to refute the hypothesis that
        atrazine by itself can adversely affect amphibian gonadal
        development and that no additional data are required to address
        this hypothesis.