2019.03.27_CCO.p14_ToxicityStudyWorkPlan

Chemours’ Proposed Toxicity Study Work Plan
Pursuant to Paragraph 14 of the Consent Order
I.IDENTIFICATION OF TEST SUBSTANCES
As stated in Attachment B to the Consent Order, the test substances identified for
mammalian and ecological toxicity testing are as follows:
Common Name Chemical Name CAS Number
PFMOAA Pefluoro-2-methoxyacetic acid 674-13-5
PFO2HXA Perfluoro(3,5-dioxahexanoic)acid 39492-88-1
PFESA-BP2/Nafion BP #2 Nafion Byproduct 2 749836-20-2
PMPA Perfluoro-2-methoxypropanoic acid 13140-29-9
PEPA 2,3,3,3-tetrafluoro-2-
(pentafluoroethoxy)propanoic acid
267239-61-2
II.IDENTIFICATION OF TESTING GUIDELINES
In order to comply with Paragraph 14 of the Consent Order, Chemours proposes to use the
following test guidelines when conducting the toxicity studies:
Toxicity Study Identified Guideline
28-day oral immunotoxicity study in rats OPPTS 870.7800
28-day oral immunotoxicity study in mice OPPTS 870.7800
90-day repeated dose oral toxicity study in rats OECD 408
90-day repeated dose oral toxicity study in mice OECD 408
Algal acute (72-hour growth) toxicity study OECD 201
Daphnid acute toxicity study OECD 202
Daphnid chronic (reproduction) toxicity study OECD 211
Fish acute toxicity study OECD 203
Sediment 10-day freshwater invertebrates toxicity test OECD 225
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III.TEST GUIDELINE SUMMARIES
A.Mammalian Studies
1. Toxicity Study: 28-day oral immunotoxicity studies in rats and mice
Guideline Summary: EPA Health Effects Test Guidelines OPPTS 870.7800 Immunotoxicity
This guideline is intended to provide information on suppression of the immune system
which might occur as a result of repeated exposure to a test chemical. In order to obtain data on
the functional responsiveness of major components of the immune system to a T cell dependent
antigen, sheep red blood cells (SRBC), rats and/or mice must be exposed to the test and control
substances for at least twenty-eight days. The animals must be immunized by intravenous or
intraperitoneal injection of SRBCs for approximately four days (depending on the strain of
animal) prior to the end of the exposure. At the end of the exposure period, either the plaque
forming cell (PFC) assay or an enzyme linked immunosorbent assay (ELISA) must be performed
to determine the effects of the test substance on the splenic anti-SRBC (IgM) response or serum
anti-SRBC IgM levels, respectively. In the event the test substance produces significant
suppression of the anti-SRBC response, expression of phenotypic markers for major lymphocyte
populations (total T and total B), and T cell subpopulations (T helpers (CD4) and T
cytotoxic/suppressors (CD8)), as assessed by flow cytometry, may be performed to determine the
effects of the test substance on either splenic or peripheral-blood lymphocyte populations and T
cell subpopulations. When this study is performed, the appropriate monoclonal antibodies for the
species being tested should be used. If the test substance has no significant effect on the anti-
SRBC assay, a functional test for NK cells may be performed to test for a chemical’s effect on
non-specific immunity. For tests performed using cells or sera from blood (ELISA or flow
cytometry), it is not necessary to destroy the animals, since immunization with SRBCs at twenty-
eight days is not expected to markedly affect the results of other assays included in sub-chronic
or longer-term studies. The necessity to perform either a quantitative analysis of the effects of a
chemical on the numbers of cells in major lymphocyte populations and T cell subpopulations by
flow cytometry, or a splenic NK cell activity assay to assess the effects of the test compound on
non-specific immunity, should be determined on a case-by-case basis, depending upon the
outcome of the anti-SRBC assay.
2. Toxicity Study: 90-day repeated dose oral toxicity study in rats and mice
Guideline Summary: OECD Test Guideline No. 408: Repeated Dose 90-Day Oral Toxicity
Study in Rodents
This guideline provides information on health hazards likely to arise from exposure to the
test substance via oral administration. The determination of sub-chronic oral toxicity using
repeated doses may be carried out after initial information on toxicity has been obtained from
acute or repeated dose 28-day toxicity tests. The method is based on the repeated oral
administration of the test substance over a prolonged period (one dose level daily during 90
days). This test guideline is intended primarily for use with rodents (preferably rats). At least 20
animals (10 female and 10 male) should be used for each test group. Three concentrations of the
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test substance, at least, should be used. The test compound is administered by gavage or via the
diet or drinking water. A limit test may be performed if no effects would be expected at a dose of
1000 mg/kg bw/d. The results of this study include: 1) measurements, including weighing at least
once a week, as well as assessment of food and water consumption; 2) daily and detailed
observations (i.e. of ophthalmological examination, haematology, clinical biochemistry and
urinalysis), preferably collected at the same time each day; and 3) gross necropsy and
histopathology. A number of endocrine-related measurements, particularly relevant to thyroid
function, have been added in 2018. A properly conducted 90-day sub-chronic test should provide
a satisfactory estimation of a no-effect level.
B.Ecotoxicological Studies
1.Algal acute (72-hour growth) toxicity study
Guideline Summary: OECD Test Guideline No. 201: Freshwater Alga and Cyanobacteria,
Growth Inhibition Test
The purpose of this test is to determine the effects of the test substance on the growth of
freshwater microalgae and/or cyanobacteria. Exponentially growing test organisms are exposed
to the test substance in batch cultures, typically over a period of 72 hours. The system response is
the reduction of growth in a series of algal cultures exposed to at least five concentrations of a
test substance. Three replicates at each test concentration should be used. The response is
evaluated as a function of the exposure concentration in comparison with the average growth of
control cultures. The cultures are allowed unrestricted exponential growth under nutrient
sufficient conditions (two alternative growth media: the OECD and the AAP) and continuous
fluorescent illumination. Growth and growth inhibition are quantified from measurements of the
algal biomass as a function of time. The limit test corresponds to one dose level of 100 mg/L.
This study includes: 1) the determination, at least daily, of the algal biomass; 2) the measure of
pH (at the beginning and at the end); and 3) microscopic observation. This test guideline
describes two response variables: average specific growth rate and yield.
2. Daphnid acute toxicity study
Guideline Summary: OECD Test Guideline No. 202: Daphnia Acute Immobilization Test
This test guideline describes an acute toxicity test to assess effects of chemicals on
daphnids (usually Daphnia magna Staus). Young daphnids, aged less than 24 hours at the start of
the test, are exposed to the test substance at a range of concentrations (at least five
concentrations) for a period of 48 hours. Immobilization is recorded at 24 hours and 48 hours
and compared with control values. The results are analyzed in order to calculate the EC50 at 48
hours. Determination of the EC50 at 24 hours is optional. At least 20 animals, preferably divided
into four groups of five animals each, should be used at each test concentration and for the
controls. At least 2 ml of test solution should be provided for each animal (i.e. a volume of 10 ml
for five daphnids per test vessel). The limit test corresponds to one dose level of 100 mg/L. The
study report should include the observation for immobilized daphnids at 24 and 48 hours after
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the beginning of the test and should also include the measures of dissolved oxygen, pH, and
concentration of the test substance at the beginning and end of the test.
3. Fish acute toxicity study
Guideline Summary: OECD Test Guideline No. 203: Fish, Acute Toxicity Test
In this test, fish are exposed to the test substance preferably for a period of 96 hours.
Mortalities are recorded at 24, 48, 72 and 96 hours and the concentrations which kill 50 percent
of the fish (LC50) are determined where possible. One or more species may be used, the choice
being at the discretion of the testing laboratory. At least seven fishes must be used at each test
concentration and in the controls. The test substance should be administered to at least five
concentrations in a geometric series with a factor preferably not exceeding 2.2. The limit test
corresponds to one dose level of 100 mg/L. This study includes the observations of fish at least
after 24, 48, 72 and 96 hours. The cumulative percentage mortality for each exposure period is
plotted against concentration on logarithmic probability paper.
4. Daphnid chronic (reproduction) toxicity study
Guideline Summary: OECD Test Guideline No. 211: Daphnia magna Reproduction Test
This test method assesses the effect of chemicals on the reproductive output of Daphnia
magna Straus. To this end, young female Daphnia are exposed to the test substance added to
water at a range of concentrations (at least five). For semi-static tests, at least 10 animals at each
test concentration and for flow-through tests, 40 animals divided into four groups of 10 animals
at each test concentration are used. The test duration is 21 days. The total number of living
offspring produced per parent animal that do not die accidentally during the test and the number
of living offspring produced per surviving parent animal at the end of the test are reported. The
study report also includes: 1) daily counting of the offspring; 2) daily recording of the parent
mortality; 3) weekly measurement of oxygen concentration, temperature, hardness and pH
values; and 4) determination of the concentrations of test substance. Optionally, other effects can
be reported, including the sex ratio of the offspring. The reproductive output of the animals
exposed to the test substance is analyzed by comparing it with that of the control in order to
determine the lowest observed effect concentration (LOEC) and hence the no observed effect
concentration (NOEC), and by estimating the concentration that causes a reduction in
reproductive output by means of a regression analysis.
5. Sediment 10-day freshwater invertebrates toxicity test
Guideline Summary: OECD Test Guideline No. 225: Sediment-Water Lumbriculus
Toxicity Test Using Spiked Sediment
This test guideline is designed to assess the effects of prolonged exposure to sediment-
associated chemicals on the reproduction and the biomass of the endobenthic oligochaete
Lumbriculus variegatus (Müller). The method is described for static test conditions. Worms of
similar physiological state are exposed to a series of toxicant concentrations applied to the
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sediment phase of a sediment-water system (artificial sediment amended with a food source and
reconstituted water). Test vessels without the test substance serve as controls. The test animals
are exposed to the sediment-water systems for a period of 28 days. The endpoints of this type of
study are the ECx for reproduction and biomass. In addition, the No Observed Effect
Concentration (NOEC), and the Lowest Observed Effect Concentration (LOEC) may be
calculated. The purpose of the study, ECx or NOEC derivation, will determine the test design. At
least five concentrations and a minimum of three replicates for ECx, or four replicates for
NOEC/LOEC, for each concentration should be tested. The test is conducted with at least 10
worms for each replicate. The report should include the total number and the dry weight of the
worms, as well as observations of abnormal behavior, mortalities, water characteristics within
the test vessels, and the total organic content.
IV.DOSE SELECTION
The amount of test substance needed for the work outlined in Attachment B to the
Consent Order is approximately 10 – 15 kg per chemical. Since none of these test substances are
commercial products, each one will require a custom synthesis followed by a detailed analysis in
order to establish the necessary levels and identify any impurities. Chemours estimates that the
synthesis and analysis will take at least one year. In order to avoid complications from batch-to-
batch inconsistencies, no toxicology studies can be performed until the full amounts are
available.
Chemours proposes a modified study design using dose levels of 0.1 mg/kg/day and 1
mg/kg/day in both rats and mice for both the 28-day and 90-day studies. The 0.1 mg/kg/day
dose corresponds to the rodent point of departure derived for the substance known as GenX in
the development of the health advisory goal of 140 ppt by the North Carolina Department of
Health and Human Services. Using these doses in the rodent studies accomplishes several goals:
•A substantial reduction (approximately 25%) in the number of rats and mice that must be
killed
•A substantial reduction in the amount of test substance required (approximately 100
grams instead of multiple kilograms)
•The ability to start the toxicology studies sooner, and thus obtain data more quickly
•Quicker development of a drinking water standard
Using this proposal, a substance that shows no effect at 0.1 mg/kg/day (the GenX rodent
point of departure) would be considered toxicologically similar to GenX itself. Therefore, the
current health advisory goal value for Gen X (140 ppt), which was developed by experts in North
Carolina based on an extensive toxicological data set, could be applied to these other substances
for which less data is available. Substances that show a significant, human-relevant toxicological
effect at a dose of 0.1 mg/kg/day would be candidates for additional work.
If no toxicity is observed at the higher dose of 1 mg/kg/day, then Chemours proposes that
1 mg/kg/day be used as a conservative point of departure for the development of a health
advisory goal. Chemours notes that the actual no-observed-adverse-effect-level could be
considerably higher (that is, less toxic) than 1 mg/kg/day, but in the interest of both a quick
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turnaround time and animal welfare, Chemours accepts 1 mg/kg/day as the point of departure in
this situation.
Using this approach will allow for a reduction in the amount of test chemical that must be
synthesized, a faster time to study start, and a reduction in the number of rodents killed, but will
still provide conservative points of departure for setting drinking water values for these
substances.
V.WORK PLAN AND SCHEDULE
The implementation of the toxicity studies will depend on the availability of the test
substances. As noted above, Chemours anticipates that it will take at least one year to complete
the synthesis and analysis for each substance (i.e. the substances are not expected to be available
before April 1, 2020). In addition, agreement with DEQ regarding the dose levels to be used for
the mammalian toxicology studies will enable a more detailed schedule to be developed.
Assuming that the contracting labs will be able to draft the appropriate protocol and obtain
approval from DEQ for the protocol within 3 months of receiving a particular test substance,
Chemours expects that the first toxicity tests will begin around July 1, 2020. The toxicity tests
for each chemical may procced once sufficient quantities are available for that particular test
substance. However, given the numerous factors that might affect the timeline of toxicity testing,
it is difficult to provide a firm start and end date for the toxicity tests discussed herein. Chemours
will continue to maintain an open dialogue with DEQ regarding the timeline for the toxicity
studies and will provide updates to the estimated timeline as soon as Chemours learns the
necessary and relevant information affecting the timeline. The chart at the end of this section
provides a summary of Chemours’ current estimated timeline.
A.Mammalian Studies
Within three months of the test substances becoming available in sufficient quantity and
quality to complete all required studies, the mammalian studies will be contracted. For the
mammalian studies, a single Contract Research Organization (CRO) that can perform all four
study types will perform the contracted work. Chemours has proposed that Charles River
Laboratories perform these studies; DEQ has approved.
The mammalian studies cannot be run in parallel because data collected in the 28-day
study will be used to refine the protocol for the 90-day study. Therefore, for each given
substance, the 28-day study will be completed prior to the start of the 90-day study. A 28-day
study typically takes 6 to 9 months to complete from the time of dosing to the issuance of the
final report. A 90-day study typically takes 9 to 12 months to complete from the time of dosing
to the issuance of a final report.
When ready, a quantity of test substance sufficient to run all four studies for that
particular substance will be shipped to the CRO. The CRO will draft protocols using the relevant
OPPT/OECD guideline as a template and submit them to DEQ for approval. Once approved by
DEQ, the study will be authorized by Chemours and the study dates will be finalized by the
CRO. Chemours will provide a copy of the final protocol and the proposed study schedule to
DEQ. Changes from the preliminary schedule resulting in significant delay will be
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communicated to DEQ. The final report will be submitted to DEQ one month after finalization
by the CRO.
B.Ecotoxicology Studies
Within three months of the test substances becoming available in sufficient quantity and
quality to complete all required studies, the ecotoxicology studies will be contracted. For the
ecotoxicological studies, a CRO that can perform all five ecotoxicology studies will be used. The
studies can be run in parallel with the exception of the Daphnid chronic (reproduction) toxicity
study (OECD 211), which is dependent on the results of the Daphnid acute toxicity study (OECD
202). Chemours has proposed that EAG Laboratories perform these studies and is awaiting
approval from DEQ.
The algal acute (72-hour growth) toxicity study (OECD 201) and the fish acute toxicity
study (OECD 203) each take approximately 6 months to complete from the time of dosing to the
report. The Daphnid acute toxicity study (OECD 202) followed by the Daphnid chronic
(reproduction) toxicity study (OECD 211) take a combined 10 months to complete
(approximately 3 months for the acute study and 7 months for the reproduction study). The
sediment 10-day freshwater invertebrates toxicity test (OECD 225) takes approximately 10
months to complete.
When ready, a quantity of test material sufficient to run all five studies for that particular
substance will be shipped to the CRO. The CRO will draft protocols using the relevant OECD
guideline as a template and submit them to DEQ for approval. Once approved by DEQ, the
studies will be authorized by Chemours and the study dates will be finalized by the CRO.
Chemours will provide a copy of the final protocol and the study schedule to DEQ. Changes
from the preliminary schedule resulting in significant delay will be communicated to DEQ. The
final report will be submitted to DEQ one month after finalization by the CRO.
C.Estimated Timeline
The chart below provides an estimated timeline for the start, duration, and completion of
the toxicity studies, as well as submission of the related final reports to DEQ. The timeline
assumes that the CRO will be able to draft its protocol, obtain approval from DEQ for the
protocol, and find capacity within its labs to begin the studies 3 months after receiving any given
test substance. Chemours reiterates that the timeline for the toxicity studies depends on many
variables that are subject to change (i.e. availability of test substance, lab capacity, approval from
DEQ). Nevertheless, the chart below reflects Chemours’ current best estimate as to the
anticipated timeline.
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Toxicity Study Estimated Study Start
(i.e. Dosing)*
Estimated CRO
Report**
Estimated Final
Report to DEQ
28-day oral
immunotoxicity study
in rats
3 months after receiving
test substance
12 months after
receiving test substance
(9 months after dosing)
13 months after
receiving test substance
28-day oral
immunotoxicity study
in mice
3 months after receiving
test substance
12 months after
receiving test substance
(9 months after dosing)
13 months after
receiving test substance
90-day repeated dose
oral toxicity study in
rats
15 months after
receiving test substance
(3 months after
completion of 28-day
study)
27 months after
receiving test substance
(12 months after
dosing)
28 months after
receiving test substance
90-day repeated dose
oral toxicity study in
mice
15 months after
receiving test substance
(3 months after
completion of 28-day
study)
27 months after
receiving test substance
(12 months after
dosing)
28 months after
receiving test substance
Algal acute (72-hour
growth) toxicity study
3 months after receiving
test substance
9 months after receiving
test substance
(6 months after dosing)
10 months after
receiving test substance
Daphnid acute toxicity
study
3 months after receiving
test substance
6 months after receiving
test substance
(3 months after dosing)
7 months after receiving
test substance
Daphnid chronic
(reproduction) toxicity
study
6 months after receiving
test substance
(upon completion of
acute study)
13 months after
receiving test substance
(7 months after dosing)
14 months after
receiving test substance
Fish acute toxicity
study
3 months after receiving
test substance
9 months after receiving
test substance
(6 months after dosing)
10 months after
receiving test substance
Sediment 10-day
freshwater invertebrates
toxicity test
3 months after receiving
test substance
13 months after
receiving test substance
(10 months after
dosing)
14 months after
receiving test substance
*The study start date depends on availability of test substance, lab capacity, approval of protocol
from DEQ, analytical capability, and other variables.
**Completion of the CRO’s report depends on dosing start date, completion of any necessary
prior studies, analytical capability, and other variables.