Chemours’ Proposed Toxicity Study Work Plan
Pursuant to Paragraph 14 of the Consent OrderI.

IDENTIFICATION OF TEST SUBSTANCESAs stated in Attachment B to the Consent Order, the test substances identified for
mammalian and ecological toxicity testing are as follows:
Common NamePFMOAAPFO2HXAPFESA-BP2/Nafion BP #2
PMPA
PEPA

II.

Chemical Name
Pefluoro-2-methoxyacetic acid
Perfluoro(3,5-dioxahexanoic)acid
Nafion Byproduct 2
Perfluoro-2-methoxypropanoic acid
2,3,3,3-tetrafluoro-2(pentafluoroethoxy)propanoic acid

CAS Number
674-13-539492-88-1
749836-20-2
13140-29-9
267239-61-2

IDENTIFICATION OF TESTING GUIDELINES

In order to comply with Paragraph 14 of the Consent Order, Chemours proposes to use thefollowing test guidelines when conducting the toxicity studies:
Toxicity Study
28-day oral immunotoxicity study in rats
28-day oral immunotoxicity study in mice
90-day repeated dose oral toxicity study in rats
90-day repeated dose oral toxicity study in mice
Algal acute (72-hour growth) toxicity study
Daphnid acute toxicity study
Daphnid chronic (reproduction) toxicity study
Fish acute toxicity study
Sediment 10-day freshwater invertebrates toxicity test

Identified Guideline
OPPTS 870.7800
OPPTS 870.7800
OECD 408
OECD 408
OECD 201
OECD 202
OECD 211
OECD 203
OECD 225

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 ofanimal) prior to the end of the exposure. At the end of the exposure period, either the plaqueforming 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 serumanti-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 theeffects 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 antiSRBC 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 twentyeight 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 achemical on the numbers of cells in major lymphocyte populations and T cell subpopulations byflow 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 theoutcome 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 thetest 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 fromacute 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 90days). 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 the2

test substance, at least, should be used. The test compound is administered by gavage or via thediet or drinking water. A limit test may be performed if no effects would be expected at a dose of1000 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 andurinalysis), preferably collected at the same time each day; and 3) gross necropsy andhistopathology. 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 exposedto 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 atest substance. Three replicates at each test concentration should be used. The response isevaluated as a function of the exposure concentration in comparison with the average growth ofcontrol 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 thealgal 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 ofpH (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 fiveconcentrations) 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 thecontrols. 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 after3

the beginning of the test and should also include the measures of dissolved oxygen, pH, andconcentration 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 percentof 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 fiveconcentrations 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 isplotted 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 towater at a range of concentrations (at least five). For semi-static tests, at least 10 animals at eachtest 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 numberof 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 canbe 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 todetermine the lowest observed effect concentration (LOEC) and hence the no observed effectconcentration (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 SedimentThis test guideline is designed to assess the effects of prolonged exposure to sedimentassociated chemicals on the reproduction and the biomass of the endobenthic oligochaete
Lumbriculus variegatus (Müller). The method is described for static test conditions. Worms ofsimilar physiological state are exposed to a series of toxicant concentrations applied to the4sediment phase of a sediment-water system (artificial sediment amended with a food source andreconstituted 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 ofstudy are the ECx for reproduction and biomass. In addition, the No Observed EffectConcentration (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 theworms, 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 theConsent Order is approximately 10 – 15 kg per chemical. Since none of these test substances arecommercial products, each one will require a custom synthesis followed by a detailed analysis inorder to establish the necessary levels and identify any impurities. Chemours estimates that thesynthesis and analysis will take at least one year. In order to avoid complications from batch-tobatch inconsistencies, no toxicology studies can be performed until the full amounts areavailable.
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
killedA 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, thecurrent 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 toxicologicaleffect 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 healthadvisory goal. Chemours notes that the actual no-observed-adverse-effect-level could beconsiderably higher (that is, less toxic) than 1 mg/kg/day, but in the interest of both a quick5

turnaround time and animal welfare, Chemours accepts 1 mg/kg/day as the point of departure inthis 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 thesesubstances.
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 forthe 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 obtainapproval 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 thenecessary 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 andquality 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 thatparticular 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 theCRO. 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
6communicated to DEQ. The final report will be submitted to DEQ one month after finalizationby the CRO.
B. Ecotoxicology Studies
Within three months of the test substances becoming available in sufficient quantity andquality 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 (OECD202). 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 thereport. 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, thestudies 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 theanticipated timeline.

7

Toxicity Study
28-day oral
immunotoxicity study
in rats
28-day oral
immunotoxicity study
in mice90-day repeated doseoral toxicity study inrats

90-day repeated doseoral toxicity study in
mice

Algal acute (72-hour
growth) toxicity study
Daphnid acute toxicity
study

Daphnid chronic(reproduction) toxicity
study
Fish acute toxicity
study
Sediment 10-day
freshwater invertebrates
toxicity test

Estimated Study Start(i.e. Dosing)*
3 months after receiving
test substance

Estimated CRO
Report**
12 months after
receiving test substance(9 months after dosing)
3 months after receiving 12 months after
test substancereceiving test substance(9 months after dosing)
15 months after
27 months after
receiving test substance receiving test substance(3 months after(12 months after
completion of 28-day
dosing)study)
15 months after
27 months after
receiving test substance receiving test substance(3 months after(12 months after
completion of 28-day
dosing)study)
3 months after receiving 9 months after receiving
test substancetest substance(6 months after dosing)
3 months after receiving 6 months after receiving
test substancetest substance(3 months after dosing)

Estimated Final
Report to DEQ
13 months after
receiving test substance

6 months after receiving
test substance(upon completion of
acute study)
3 months after receiving
test substance

13 months after
receiving test substance(7 months after dosing)

14 months after
receiving test substance

9 months after receiving
test substance(6 months after dosing)
3 months after receiving 13 months after
test substancereceiving test substance(10 months after
dosing)

10 months after
receiving test substance

13 months after
receiving test substance
28 months after
receiving test substance

28 months after
receiving test substance

10 months after
receiving test substance
7 months after receiving
test substance

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 necessaryprior studies, analytical capability, and other variables.

8