Draft Risk Evaluation for Tris(2-chloroethyl) phosphate (TCEP) Systematic Review Supplemental File: Data Quality Evaluation Information for Environmental Hazard CASRN: 115-96-8


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December 2023

SEPA

December 2023

United States	Office of Chemical Safety and

Environmental Protection Agency	Pollution Prevention

Draft Risk Evaluation for
Tris(2-chloroethyl) phosphate (TCEP)

Systematic Review Supplemental File:

Data Quality Evaluation Information for
Environmental Hazard

CASRN: 115-96-8

December 2023

-------
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This supplemental file contains information regarding the data quality evaluation results relevant to the characterization
of environmental hazard for the Draft Risk Evaluation for Tris(2-chloroethyl) phosphate (TCEP). EPA conducted data quality
evaluation based on author-reported descriptions and results; additional analyses (e.g., statistical analyses performed during
data integration into the risk evaluation) potentially conducted by EPA are not contained in this supplemental file. EPA used
the TSCA systematic review process described in the Draft Systematic Review Protocol Supporting TSCA Risk Evaluations
for Chemical Substances (also referred to as '2021 Draft Systematic Review Protocol'). Any updated steps in the systematic
review process since the publication of the 2021 Draft Systematic Review Protocol are described in the Draft Risk Evaluation
for Tris(2-chloroethyl) Phosphate (TCEP) - Systematic Review Protocol.

Different data quality evaluation forms were used depending on the organism as described in the PECO statement in Ap-
pendix H.5.7 of the 2021 Draft Systematic Review Protocol. Each health outcome was evaluated independently within a given
reference, therefore each reference may have more than one overall quality determination (OQD) to more appropriately reflect
the quality of each health outcome and the respective hazard endpoints as described by the study authors. Some data evalua-
tion forms have general additional comments presented adjacent to the OQD to add further context. No OQD is determined
for each reference as a whole, if it contains data from more than one evidence stream. The table of contents lists references
based on chemical, broad habitat (e.g., aquatic, terrestrial), taxa, taxonomic group, exposure duration, and health outcome (e.g.,
mortality) categories relevant to the endpoint being evaluated.

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Table of Contents

Table of Contents

HERO ID

Reference

Page

Tris(2-chloroethyl) phosphate (TCEP)

Habitat: Aquatic (freshwater)

Taxa: Vertebrates

Danio rerio

5164137

Alzualde, A., Behl, M., Sipes, N. S., Hsieh, J. H., Alday, A., Tice, R. R., Paules, R. S., Muriana, A., Quevedo, C. (2018). Toxicity
profiling of flame retardants in zebrafish embryos using a battery of assays for developmental toxicity, neurotoxicity, cardiotoxicity and
hepatotoxicity toward human relevance. Neurotoxicology and Teratology 70:40-50.

3014520

Dishaw, L. V., Hunter, D. L., Padnos, B., Padilla, S., Stapleton, H. M. (2014). Developmental Exposure to Organophosphate Flame
Retardants Elicits Overt Toxicity and Alters Behavior in Early Life Stage Zebrafish (Danio rerio). Toxicological Sciences 142(2):445-454.

18

4290535

4180931

Du, Z., Wang, G., Gao, S., Wang, Z. (2015). Aryl organophosphate flame retardants induced cardiotoxicity during zebrafish embryogenesis:
by disturbing expression of the transcriptional regulators. Aquatic Toxicology 161:25-32.

Jarema, K. A., Hunter, D. L., Shaffer, R. M., Behl, M., Padilla, S. (2015). Acute and developmental behavioral effects of flame retardants
and related chemicals in zebrafish. Neurotoxicology and Teratology 52(Pt B):194-209.

20

24

7274629

Lee, J. S., Morita, Y., Kawai, Y. K., Covaci, A., Kubota, A. (2020). Developmental circulatory failure caused by metabolites of organophos-
phorus flame retardants in zebrafish, Danio rerio. Chemosphere 246:125738.

26

2953504

Noyes, P. D., Haggard, D. E., Gonnerman, G. D., Tanguay, R. L. (2015). Advanced morphological - behavioral test platform reveals
neurodevelopmental defects in embryonic zebrafish exposed to comprehensive suite of halogenated and organophosphate flame retardants.
Toxicological Sciences 145(1):177-195.

32

5166352

Wang, G. W., Chen, H. Y., Du, Z. K., Li, J. H., Wang, Z. Y., Gao, S. X. (2017). In vivo metabolism of organophosphate flame retardants
and distribution of their main metabolites in adult zebrafish. Science of the Total Environment 590:50-59.

40

3479540

Behl, M., Hsieh, J. H., Shafer, T. J., Mundy, W. R., Rice, J. R., Boyd, W. A., Freedman, J. H., Hunter, E. S., Jarema, K. A., Padilla, S.,
Tice, R. R. (2015). Use of alternative assays to identify and prioritize organophosphorus flame retardants for potential developmental and
neurotoxicity. Neurotoxicology and Teratology 52(Pt B):181-193.

42

3014520

Dishaw, L. V., Hunter, D. L., Padnos, B., Padilla, S., Stapleton, H. M. (2014). Developmental Exposure to Organophosphate Flame
Retardants Elicits Overt Toxicity and Alters Behavior in Early Life Stage Zebrafish (Danio rerio). Toxicological Sciences 142(2):445-454.

46

4180931

Jarema, K. A., Hunter, D. L., Shaffer, R. M., Behl, M., Padilla, S. (2015). Acute and developmental behavioral effects of flame retardants
and related chemicals in zebrafish. Neurotoxicology and Teratology 52(Pt B):194-209.

54

5469290

Li, R., Wang, H., Mi, C., Feng, C., Zhang, L., Yang, L., Zhou, B. (2019). The adverse effect of TCIPP and TCEP on neurodevelopment of
zebrafish embryos/larvae. Chemosphere 220:811-817.

56

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Table of Contents

2953504

Noyes, P. D., Haggard, D. E., Gonnerman, G. D., Tanguay, R. L. (2015). Advanced morphological - behavioral test platform reveals
neurodevelopmental defects in embryonic zebrafish exposed to comprehensive suite of halogenated and organophosphate flame retardants.
Toxicological Sciences 145(1):177-195.

66

5469203

Sun, L., Xu, W., Peng, T.,ao, Chen, H., Ren, L.,in, Tan, H., Xiao, D.,an, Qian, H., Fu, Z. (2016). Developmental exposure of zebrafish
larvae to organophosphate flame retardants causes neurotoxicity. Neurotoxicology and Teratology 55:16-22.

75

5469243

Wu, Y., Su, G., Tang, S., Liu, W., Ma, Z., Zheng, X., Liu, H., Yu, H. (2017). The combination of in silico and in vivo approaches for the
investigation of disrupting effects of tris (2-chloroethyl) phosphate (TCEP) toward core receptors of zebrafish. Chemosphere 168:122-130.

79

Oryzias latipes

4292102

Sun, L., Tan, H., Peng, T., Wang, S., Xu, W., Qian, H., Jin, Y., Fu, Z. (2016). Developmental neurotoxicity of organophosphate flame
retardants in early life stages of Japanese medaka (Oryzias latipes). Environmental Toxicology and Chemistry 35(12):2931-2940.

89

Salmo gairdneri

6310866

Life Sciences Research Ltd, (1990). Fyrol CEF: Acute toxicity to rainbow trout.

99

Salmo salar

5469341

Arukwe, A., Carteny, C. C., Eggen, T. (2016). Lipid peroxidation and oxidative stress responses in juvenile salmon exposed to waterborne
levels of the organophosphate compounds tris(2-butoxyethyl)- and tris(2-chloroethyl) phosphates. Journal of Toxicology and Environmen-
tal Health, Part A: Current Issues 79(13-15):515-525.

103

Taxa: Invertebrates

Daphnia magna

5184752

Kovacevic, V., Simpson, A. J., Simpson, M. J. (2018). Investigation of daphnia magna sub-lethal exposure to organophosphate esters in the
presence of dissolved organic matter using "H NMR-based metabolomics. Metabolites 8(2):34.

105

Dugesia japonica

10064285

Zhang, S., Hagstrom, D., Hayes, P., Graham, A., Collins, E. S. (2019). Multi-behavioral endpoint testing of an 87-chemical compound
library in freshwater planarians. Toxicological Sciences 167(l):26-44.

107

5469417

Zhang, S., Ireland, D., Sipes, N. S., Behl, M., Collins, E. S. (2019). Screening for neurotoxic potential of 15 flame retardants using
freshwater planarians. Neurotoxicology and Teratology 73:54-66.

119

Habitat: Terrestrial

Taxa: Vertebrates

Falco sparverius

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Table of Contents

5353113

Fernie, K. J., Palace, V., Peters, L. E., Basu, N., Letcher, R. J., Karouna-Renier, N. K., Schultz, S. L., Lazarus, R. S., Rattner, B. A.
(2015). Investigating endocrine and physiological parameters of captive American kestrels exposed by diet to selected organophosphate
flame retardants. Environmental Science and Technology 49(12):7448-7455.

137

Gallus gallus domesticus

5165206

Stauffer Chem Co, (1981). Toxicology reports on FYROL FR-2 (volume I - II) with attachments and cover letter dated 020381. nan
8100271:#88-8100271.

143

Taxa: Invertebrates

Caenorhabditis elegans

3479540

Behl, M., Hsieh, J. H., Shafer, T. J., Mundy, W. R., Rice, J. R., Boyd, W. A., Freedman, J. H., Hunter, E. S., Jarema, K. A., Padilla, S.,
Tice, R. R. (2015). Use of alternative assays to identify and prioritize organophosphorus flame retardants for potential developmental and
neurotoxicity. Neurotoxicology and Teratology 52(Pt B):181-193.

145

3975281

Behl, M., Rice, J. R., Smith, M. V., Co, C. A., Bridge, M. F., Hsieh, J. H., Freedman, J. H., Boyd, W. A. (2016). Editor's highlight:
Comparative toxicity of organophosphate flame retardants and polybrominated diphenyl ethers to Caenorhabditis elegans. Toxicological
Sciences 154(2):241-252.

147

5469475

Xu, T., Li, P., Wu, S., Lei, L., He, D. (2017). Tris(2-chloroethyl) phosphate (TCEP) and tris(2-chloropropyl) phosphate (TCPP) induce
locomotor deficits and dopaminergic degeneration in Caenorhabditis elegans. Toxicology Research 6(l):63-72.

153

Eisenia fetida

5469239

Yang, Y., Xiao, Y., Chang, Y., Cui, Y., Klobucar, G., Li, M. (2018). Intestinal damage, neurotoxicity and biochemical responses caused by
tris (2-chloroethyl) phosphate and tricresyl phosphate on earthworm. Ecotoxicology and Environmental Safety 158:78-86.

163

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Tris(2-chloroethyl) phosphate (TCEP) Environmental Hazard Evaluation HERO ID: 5164137 Table: 1 of 6

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Alzualde, A., Behl, M., Sipes, N. S., Hsieh, J. H., Alday, A., Tice, R. R., Paules, R. S., Muriana, A., Quevedo, C. (2018). Toxicity profiling of flame retar-
dants in zebrafish embryos using a battery of assays for developmental toxicity neurotoxicity cardiotoxicity and hepatotoxicity toward human relevance.
Neurotoxicology and Teratology 7040-50.

Overall Duration: 0 - 4 days (0-96h); Exposure Duration: 0 - 4 days (0-96h)

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ Embryo
Development/Growth
Tris(2-chloroethyl) phosphate (TCEP)

5164137

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1: Test Substance Identity
Metric 2: Test Substance Source
Metric 3: Test Substance Purity

High Tris(2-chloroethyl) phosphate (TCEP), CASRN 115-96-8
High Supplied by Sigma-Aldrich, Lot number SZBE090XV
High Purity = 95.6%

Domain 2: Test Design

Metric 4: Negative Controls High Vehicle/Solvent control (0.5% DMSO) and positive controls were used. No negative

control reported. No mortality was reported in the vehicle control so lack of negative
control is expected to have minimal impact on the results.

Metric 5: Negative Control Response Low Biological response was difficult to interpret. Figure 1 reported affected embryos, but

not for TCEP and based on other examples, "affected embryos" was near 10% with no
mortality reported.

Metric 6: Randomized Allocation Low Organism allocation was not reported.

Domain 3: Exposure Characterization

Metric 7:

Experimental System/Test Media

Medium

Study used well plates for experiments. Covering of well plates were not reported. Ex-

Preparation

posure solutions were renewed on day two. However, remaining concentration in well

plate prior to renewal was not reported.

Metric 8:

Consistency of Exposure

High

Exposures were consistent across groups.

Administration

Metric 9:

Measurement of Test Substance

Medium

Exposure solutions were renewed on day two. However, remaining concentration in well

Concentration

plate prior to renewal, and end of test concentration on day four were not reported.

Metric 10:

Exposure Duration and Frequency

High

Duration and frequency were appropriate for the test.

Metric 11:

Number of Exposure Groups/

High

Exposure group dosage and spacing were appropriate following a dose-range finding

Spacing of Exposure Levels

study.

Metric 12:

Testing at or Below Solubility Limit

High

No indications were reported that TCEP concentration were above solubility limit. The

study reported precipitation of other chemicals tested.

Domain 4: Test Organism

Metric 13: Test Organism Characteristics High

Metric 14: Acclimatization and Pretreatment Low

Conditions

Embryos and life stages were adequately described.
Study did not report an acclimation period.

Continued on next page ...

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Environmental Hazard Evaluation

HERO ID: 5164137 Table: 1 of 6

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Overall Duration: 0 - 4 days (0-96h); Exposure Duration: 0 - 4 days (0-96h)

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ Embryo
Development/Growth
Tris(2-chloroethyl) phosphate (TCEP)

5164137

Domain

Metric

Rating

Comments

Metric 15:

Number of Organisms and
Replicates per Group

Low

Number of replicates were not reported.

Domain 5: Outcome Assessment

Metric 16:
Metric 17:
Metric 18:

Adequacy of Test Conditions
Outcome Assessment Methodology
Consistency of Outcome
Assessment.

High
High
High

Housing and environment were appropriate for the test.
Intended outcomes were reported.

Assessment were consistent among groups.

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure

High No confounding variable were reported.

High Outcomes unrelated to exposure were not reported.

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods

Metric 22: Reporting of Data

Metric 23: Explanation of Unexpected Outcomes

High Percentage of altered and dead embryos wasused for Effective Concentration 50%
(EC50) and Lethal Concentration 50% (LC50) calculations applying a nonlinear re-
gression test (sigmoidal dose-response curve) using the GraphPad Prism (GraphPad
Software).

Low Data were not clearly presented or described, but calculated endpoints were presented in
the text and discussed.

High No unexpected outcomes were reported.

Additional Comments: None

Overall Quality Determination

High

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Environmental Hazard Evaluation

HERO ID: 5164137 Table: 2 of 6

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Overall Duration: 0 - 4 days (0-96h); Exposure Duration: 0 - 4 days (0-96h)

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ Larvae
Behavioral

Tris(2-chloroethyl) phosphate (TCEP)

5164137

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1: Test Substance Identity
Metric 2: Test Substance Source
Metric 3: Test Substance Purity

High Tris(2-chloroethyl) phosphate (TCEP), CASRN 115-96-8
High Supplied by Sigma-Aldrich, Lot number SZBE090XV
High Purity = 95.6%

Domain 2: Test Design

Metric 4: Negative Controls High Vehicle/Solvent control (0.5% DMSO) and positive controls were used. No negative

control reported. No mortality was reported in the vehicle control so lack of negative
control is expected to have minimal impact on the results.

Metric 5: Negative Control Response Low Biological response was difficult to interpret. Figure 1 reported mortality, but not for

TCEP and based on other examples, "affected embryos" was near 10% with no mortality
reported.

Metric 6: Randomized Allocation Low Organism allocation was not reported.

Domain 3: Exposure Characterization

Metric 7:

Experimental System/Test Media

Medium

Study used well plates for experiments. Covering of well plates were not reported. Ex-

Preparation

posure solutions were renewed on day two. However, remaining concentration in well

plate prior to renewal was not reported.

Metric 8:

Consistency of Exposure

High

Exposures were consistent across groups.

Administration

Metric 9:

Measurement of Test Substance

Low

Only initial nominal exposure concentrations were reported.

Concentration

Metric 10:

Exposure Duration and Frequency

High

Duration and frequency were appropriate for the test.

Metric 11:

Number of Exposure Groups/

High

Exposure group dosage and spacing were appropriate following a dose-range finding

Spacing of Exposure Levels

study.

Metric 12:

Testing at or Below Solubility Limit

High

No indications were reported that TCEP concentration were above solubility limit. The

study reported precipitation of other chemicals tested.

Domain 4: Test Organism

Metric 13:
Metric 14:

Metric 15:

Test Organism Characteristics High

Acclimatization and Pretreatment Low

Conditions

Number of Organisms and Low
Replicates per Group

Embryos and life stages were adequately described.

10 min acclimation period was reported prior to behavior assessment (locomotor activ-
ity).

Number of replicates were not reported.

Continued on next page ...

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Environmental Hazard Evaluation

HERO ID: 5164137 Table: 2 of 6

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Overall Duration: 0 - 4 days (0-96h); Exposure Duration: 0 - 4 days (0-96h)

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ Larvae
Behavioral

Tris(2-chloroethyl) phosphate (TCEP)

5164137

Domain

Metric

Rating

Comments

Domain 5: Outcome Assessment

Metric 16:

Adequacy of Test Conditions

High

Housing and environment were appropriate for the test.

Metric 17:

Outcome Assessment Methodology

High

Intended outcomes were reported.

Metric 18:

Consistency of Outcome

High

Assessment were consistent among groups.

Assessment.

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test High

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure High

No confounding variable were reported.

Outcomes unrelated to exposure were not reported.

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods High The mean of the total distance moved by embryos in each group asmeasured in two-

minute time bins and treated versus control groups were compared using unpaired Stu-
dent's t-test.

Metric 22: Reporting of Data Low Data were not clearly presented or described, but calculated endpoints were presented in

the text and discussed.

Metric 23: Explanation of Unexpected Outcomes High No unexpected outcomes were reported.

Additional Comments: None

Overall Quality Determination High

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Tris(2-chloroethyl) phosphate (TCEP) Environmental Hazard Evaluation HERO ID: 5164137 Table: 3 of 6

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Overall Duration: 0 - 4 days (0-96h); Exposure Duration: 0 - 4 days (0-96h)

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ Embryo
Hepatic/Liver

Tris(2-chloroethyl) phosphate (TCEP)

5164137

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1: Test Substance Identity
Metric 2: Test Substance Source
Metric 3: Test Substance Purity

High Tris(2-chloroethyl) phosphate (TCEP), CASRN 115-96-8
High Supplied by Sigma-Aldrich, Lot number SZBE090XV
High Purity = 95.6%

Domain 2: Test Design

Metric 4: Negative Controls High Vehicle/Solvent control (0.5% DMSO) and positive controls were used. No negative

control reported. No mortality was reported in the vehicle control so lack of negative
control is expected to have minimal impact on the results.

Metric 5: Negative Control Response High Biological response was difficult to interpret. Figure 1 reported affected embryos, but

not for TCEP and based on other examples, "affected embryos" was near 10% with no
mortality reported.

Metric 6: Randomized Allocation Low Organism allocation was not reported.

Domain 3: Exposure Characterization

Metric 7:

Experimental System/Test Media

Medium

Study used well plates for experiments. Covering of well plates were not reported. Ex-

Preparation

posure solutions were renewed on day two. However, remaining concentration in well

plate prior to renewal was not reported.

Metric 8:

Consistency of Exposure

High

Exposures were consistent across groups.

Administration

Metric 9:

Measurement of Test Substance

Medium

Exposure solutions were renewed on day two. However, remaining concentration in well

Concentration

plate prior to renewal, and end of test concentration on day four were not reported.

Metric 10:

Exposure Duration and Frequency

High

Duration and frequency were appropriate for the test.

Metric 11:

Number of Exposure Groups/

High

Exposure group dosage and spacing were appropriate following a dose-range finding

Spacing of Exposure Levels

study.

Metric 12:

Testing at or Below Solubility Limit

High

No indications were reported that TCEP concentration were above solubility limit. The

study reported precipitation of other chemicals tested.

Domain 4: Test Organism

Metric 13: Test Organism Characteristics High Embryos and life stages were adequately described.

Metric 14: Acclimatization and Pretreatment Low Study did not report an acclimation period.

Conditions

Metric 15: Number of Organisms and Low Number of replicates were not reported.
Replicates per Group

Continued on next page ...

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Environmental Hazard Evaluation

HERO ID: 5164137 Table: 3 of 6

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Overall Duration: 0 - 4 days (0-96h); Exposure Duration: 0 - 4 days (0-96h)

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ Embryo
Hepatic/Liver

Tris(2-chloroethyl) phosphate (TCEP)

5164137

Domain

Metric

Rating

Comments

Domain 5: Outcome Assessment

Metric 16:

Adequacy of Test Conditions

High

Housing and environment were appropriate for the test.

Metric 17:

Outcome Assessment Methodology

High

Intended outcomes were reported.

Metric 18:

Consistency of Outcome

High

Assessment were consistent among groups.

Assessment.

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test High

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure High

No confounding variable were reported.

Outcomes unrelated to exposure were not reported.

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods

Metric 22: Reporting of Data

Metric 23: Explanation of Unexpected Outcomes

Low Percentage of altered and dead embryos was used for Effective Concentration 50%
(EC50) and Lethal Concentration 50% (LC50) calculations applying a nonlinear re-
gression test (sigmoidal dose-response curve) using the GraphPad Prism (GraphPad
Software).

High Data were not clearly presented or described, but calculated endpoints were presented in
the text and discussed.

High No unexpected outcomes were reported.

Additional Comments: None

Overall Quality Determination

High

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Tris(2-chloroethyl) phosphate (TCEP) Environmental Hazard Evaluation HERO ID: 5164137 Table: 4 of 6

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Overall Duration: 0 - 4 days (0-96h); Exposure Duration: 0 - 4 days (0-96h)

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ Embryo
Cardiovascular

Tris(2-chloroethyl) phosphate (TCEP)

5164137

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1: Test Substance Identity
Metric 2: Test Substance Source
Metric 3: Test Substance Purity

High Tris(2-chloroethyl) phosphate (TCEP), CASRN 115-96-8
High Supplied by Sigma-Aldrich, Lot number SZBE090XV
High Purity = 95.6%

Domain 2: Test Design

Metric 4: Negative Controls High Vehicle/Solvent control (0.5% DMSO) and positive controls were used. No negative

control reported. No mortality was reported in the vehicle control so lack of negative
control is expected to have minimal impact on the results.

Metric 5: Negative Control Response High Biological response was difficult to interpret. Figure 1 reported affected embryos, but

not for TCEP and based on other examples, "affected embryos" was near 10% with no
mortality reported.

Metric 6: Randomized Allocation Low Organism allocation was not reported.

Domain 3: Exposure Characterization

Metric 7:

Experimental System/Test Media

Medium

Study used well plates for experiments. Covering of well plates were not reported. Ex-

Preparation

posure solutions were renewed on day two. However, remaining concentration in well

plate prior to renewal was not reported.

Metric 8:

Consistency of Exposure

High

Exposures were consistent across groups.

Administration

Metric 9:

Measurement of Test Substance

Medium

Exposure solutions were renewed on day two. However, remaining concentration in well

Concentration

plate prior to renewal, and end of test concentration on day four were not reported.

Metric 10:

Exposure Duration and Frequency

High

Duration and frequency were appropriate for the test.

Metric 11:

Number of Exposure Groups/

High

Exposure group dosage and spacing were appropriate following a dose-range finding

Spacing of Exposure Levels

study.

Metric 12:

Testing at or Below Solubility Limit

High

No indications were reported that TCEP concentration were above solubility limit. The

study reported precipitation of other chemicals tested.

Domain 4: Test Organism

Metric 13: Test Organism Characteristics High Embryos and life stages were adequately described.

Metric 14: Acclimatization and Pretreatment Low Study did not report an acclimation period.

Conditions

Metric 15: Number of Organisms and Low Number of replicates were not reported.
Replicates per Group

Continued on next page ...

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5164137 Table: 4 of 6

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Overall Duration: 0 - 4 days (0-96h); Exposure Duration: 0 - 4 days (0-96h)

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ Embryo
Cardiovascular

Tris(2-chloroethyl) phosphate (TCEP)

5164137

Domain

Metric

Rating

Comments

Domain 5: Outcome Assessment

Metric 16:

Adequacy of Test Conditions

High

Housing and environment were appropriate for the test.

Metric 17:

Outcome Assessment Methodology

High

Intended outcomes were reported.

Metric 18:

Consistency of Outcome

High

Assessment were consistent among groups.

Assessment.

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test High

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure High

No confounding variable were reported.

Outcomes unrelated to exposure were not reported.

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods

Metric 22: Reporting of Data

Metric 23: Explanation of Unexpected Outcomes

High Data were not clearly presented or described, but calculated endpoints were presented in
the text and discussed.

High No unexpected outcomes were reported.

Additional Comments: None

Overall Quality Determination

High

Page 13 of 170

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December 2023

Tris(2-chloroethyl) phosphate (TCEP) Environmental Hazard Evaluation HERO ID: 5164137 Table: 5 of 6

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Overall Duration: 0 - 4 days (0-96h); Exposure Duration: 0 - 4 days (0-96h)

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ Embryo
ADME (biotransformation)

Tris(2-chloroethyl) phosphate (TCEP)

5164137

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1: Test Substance Identity
Metric 2: Test Substance Source
Metric 3: Test Substance Purity

High Tris(2-chloroethyl) phosphate (TCEP), CASRN 115-96-8
High Supplied by Sigma-Aldrich, Lot number SZBE090XV
High Purity = 95.6%

Domain 2: Test Design

Metric 4: Negative Controls High Vehicle/Solvent control (0.5% DMSO) and positive controls were used. No negative

control reported. No mortality was reported in the vehicle control so lack of negative
control is expected to have minimal impact on the results.

Metric 5: Negative Control Response High Biological response was difficult to interpret. Figure 1 reported affected embryos, but

not for TCEP and based on other examples, "affected embryos" was near 10% with no
mortality reported.

Metric 6: Randomized Allocation Low Organism allocation was not reported.

Domain 3: Exposure Characterization

Metric 7:

Experimental System/Test Media

Medium

Study used well plates for experiments. Covering of well plates were not reported. Ex-

Preparation

posure solutions were renewed on day two. However, remaining concentration in well

plate prior to renewal was not reported.

Metric 8:

Consistency of Exposure

High

Exposures were consistent across groups.

Administration

Metric 9:

Measurement of Test Substance

Medium

Exposure solutions were renewed on day two. However, remaining concentration in well

Concentration

plate prior to renewal, and end of test concentration on day four were not reported.

Metric 10:

Exposure Duration and Frequency

High

Duration and frequency were appropriate for the test.

Metric 11:

Number of Exposure Groups/

High

Exposure group dosage and spacing were appropriate following a dose-range finding

Spacing of Exposure Levels

study.

Metric 12:

Testing at or Below Solubility Limit

High

No indications were reported that TCEP concentration were above solubility limit. The

study reported precipitation of other chemicals tested.

Domain 4: Test Organism

Metric 13: Test Organism Characteristics High Embryos and life stages were adequately described.

Metric 14: Acclimatization and Pretreatment Low Study did not report an acclimation period.

Conditions

Metric 15: Number of Organisms and Low Number of replicates were not reported.
Replicates per Group

Continued on next page ...

Page 14 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5164137 Table: 5 of 6

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Overall Duration: 0 - 4 days (0-96h); Exposure Duration: 0 - 4 days (0-96h)

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ Embryo
ADME (biotransformation)

Tris(2-chloroethyl) phosphate (TCEP)

5164137

Domain

Metric

Rating

Comments

Domain 5: Outcome Assessment

Metric 16:
Metric 17:
Metric 18:

Adequacy of Test Conditions High
Outcome Assessment Methodology High
Consistency of Outcome High
Assessment.

Housing and environment were appropriate for the test.
Intended outcomes were reported.

Assessment were consistent among groups.

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test High

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure High

No confounding variable were reported.

Outcomes unrelated to exposure were not reported.

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods

Metric 22: Reporting of Data

Metric 23: Explanation of Unexpected Outcomes

High Data were not clearly presented or described, but calculated endpoints were presented in
the text and discussed.

High No unexpected outcomes were reported.

Additional Comments: None

Overall Quality Determination

High

Page 15 of 170

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December 2023

Tris(2-chloroethyl) phosphate (TCEP) Environmental Hazard Evaluation HERO ID: 5164137 Table: 6 of 6

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Overall Duration: 0 - 4 days (0-96h); Exposure Duration: 0 - 4 days (0-96h)

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ Embryo
Mortality

Tris(2-chloroethyl) phosphate (TCEP)

5164137

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1: Test Substance Identity
Metric 2: Test Substance Source
Metric 3: Test Substance Purity

High Tris(2-chloroethyl) phosphate (TCEP), CASRN 115-96-8
High Supplied by Sigma-Aldrich, Lot number SZBE090XV
High Purity = 95.6%

Domain 2: Test Design

Metric 4: Negative Controls High Vehicle/Solvent control (0.5% DMSO) and positive controls were used. No negative

control reported. No mortality was reported in the vehicle control so lack of negative
control is expected to have minimal impact on the results.

Metric 5: Negative Control Response High Biological response was difficult to interpret. Figure 1 reported affected embryos, but

not for TCEP and based on other examples, "affected embryos" was near 10% with no
mortality reported.

Metric 6: Randomized Allocation Low Organism allocation was not reported.

Domain 3: Exposure Characterization

Metric 7:

Experimental System/Test Media

Medium

Study used well plates for experiments. Covering of well plates were not reported. Ex-

Preparation

posure solutions were renewed on day two. However, remaining concentration in well

plate prior to renewal was not reported.

Metric 8:

Consistency of Exposure

High

Exposures were consistent across groups.

Administration

Metric 9:

Measurement of Test Substance

Medium

Exposure solutions were renewed on day two. However, remaining concentration in well

Concentration

plate prior to renewal, and end of test concentration on day four were not reported.

Metric 10:

Exposure Duration and Frequency

High

Duration and frequency were appropriate for the test.

Metric 11:

Number of Exposure Groups/

High

Exposure group dosage and spacing were appropriate following a dose-range finding

Spacing of Exposure Levels

study.

Metric 12:

Testing at or Below Solubility Limit

High

No indications were reported that TCEP concentration were above solubility limit. The

study reported precipitation of other chemicals tested.

Domain 4: Test Organism

Metric 13: Test Organism Characteristics High Embryos and life stages were adequately described.

Metric 14: Acclimatization and Pretreatment Low Study did not report an acclimation period.

Conditions

Metric 15: Number of Organisms and Low Number of replicates were not reported.
Replicates per Group

Continued on next page ...

Page 16 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5164137 Table: 6 of 6

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Overall Duration: 0 - 4 days (0-96h); Exposure Duration: 0 - 4 days (0-96h)

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ Embryo
Mortality

Tris(2-chloroethyl) phosphate (TCEP)

5164137

Domain

Metric

Rating

Comments

Domain 5: Outcome Assessment

Metric 16:

Adequacy of Test Conditions

High

Housing and environment were appropriate for the test.

Metric 17:

Outcome Assessment Methodology

High

Intended outcomes were reported.

Metric 18:

Consistency of Outcome

High

Assessment were consistent among groups.

Assessment.

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test High

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure High

No confounding variable were reported.

Outcomes unrelated to exposure were not reported.

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods

Metric 22: Reporting of Data

Metric 23: Explanation of Unexpected Outcomes

High Data were not clearly presented or described, but calculated endpoints were presented in
the text and discussed.

High No unexpected outcomes were reported.

Additional Comments: None

Overall Quality Determination

High

Page 17 of 170

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December 2023

Tris(2-chloroethyl) phosphate (TCEP) Environmental Hazard Evaluation HERO ID: 3014520 Table: 1 of 1

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Dishaw, L. V., Hunter, D. L., Padnos, B., Padilla, S., Stapleton, H. M. (2014). Developmental Exposure to Organophosphate Flame Retardants Elicits Overt
Toxicity and Alters Behavior in Early Life Stage Zebraflsh (Danio rerio). Toxicological Sciences 142(2):445-454.

Overall Duration: 0 - 4 days (0-96h); Exposure Duration: 0 - 4 days (0-96h)

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ Embryo
Nutritional and Metabolic
Tris(2-chloroethyl) phosphate (TCEP)

3014520

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1: Test Substance Identity
Metric 2: Test Substance Source

Metric 3: Test Substance Purity

High
High

High

The chemical substance was identified as the organophosphate flame retardant, TCEP.
The test substance was obtained from a reputable chemical manufacturer. The deuter-
ated TCEP internal standards were synthesized in a single lab.

The test substance had a 97% purity.

Domain 2: Test Design

Metric 4: Negative Controls Uninformative The metabolism study compared 1 day post fertilization and 5 day post fertilization

parent chemical to metabolites in embryos and larvae. No negative control reported.

Metric 5: Negative Control Response Uninformative The study evaluated parent chemical to metabolite at 24 hours in embryos to levels in

larvae at 5 days exposure. No negative control was used.

Metric 6: Randomized Allocation Low embryos were viewed under a dissecting microscope and only healthy embryos with

intact chorions were selected for use in experiments. This is a supplemental metabolite
study in zebraflsh embryos.

Domain 3: Exposure Characterization

Metric 7:

Experimental System/Test Media

High

The experimental system and methods for preparation of test media were described in

Preparation

adequate detail.

Metric 8:

Consistency of Exposure

High

Details were sufficient to indicate consistency of exposure over 24 hour period for em-

Administration

bryos.

Metric 9:

Measurement of Test Substance

Medium

Embryos were exposed to 1 uM solution of TCEP (in 10% Hanks' balanced salt solu-

Concentration

tion) in glass petri dishes for 24 hours.

Metric 10:

Exposure Duration and Frequency

High

The 24-hour exposure was appropriate for this metabolism study.

Metric 11:

Number of Exposure Groups/

High

Exposures were to a single concentration in embryos to evaluate metabolism, which was

Spacing of Exposure Levels

later compared with 5-day old larva metabolism.

Metric 12:

Testing at or Below Solubility Limit

High

This was a metabolism study with a single concentration (which was below the water

solubility limit of TCEP).

Domain 4: Test Organism

Metric 13: Test Organism Characteristics

Metric 14: Acclimatization and Pretreatment
Conditions

Low
Low

The test species is an established model for toxicity testing, but the authors did not re-
port the the source of the zebraflsh used in the study.

Controls were not used for the metabolism study, and the authors referenced Padilla et
al., 2011 for details on acclimatization and pretreatment.

Continued on next page .

Page 18 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 3014520 Table: 1 of 1

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Dishaw, L. V., Hunter, D. L., Padnos, B., Padilla, S., Stapleton, H. M. (2014). Developmental Exposure to Organophosphate Flame Retardants Elicits Overt
Toxicity and Alters Behavior in Early Life Stage Zebrafish (Danio rerio). Toxicological Sciences 142(2):445-454.

Overall Duration: 0 - 4 days (0-96h); Exposure Duration: 0 - 4 days (0-96h)

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ Embryo
Nutritional and Metabolic
Tris(2-chloroethyl) phosphate (TCEP)

3014520

Domain

Metric

Rating

Comments

Metric 15:

Number of Organisms and
Replicates per Group

Medium

This embryo metabolism test used 75-95 embryos per petri dish with 3 replicates.

Domain 5: Outcome Assessment

Metric 16:

Metric 17:
Metric 18:

Adequacy of Test Conditions

Outcome Assessment Methodology
Consistency of Outcome
Assessment.

High

High
High

The embryos were collected from breeding tanks, washed and bleached, and healthy
embryos were maintained in glass petri dishes maintained at 26C.

The assessment methodology assessed metabolism after 24 hours of exposure.
Outcomes were assessed consistently across exposure groups.

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure

High
High

The authors did not report any variations in environmental conditions or other factors
that could impact results.

The authors selected for healthy, viable embryos so that there were no differences
among groups that could influence the outcome assessment.

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods High Tissue concentrations of the parent and primary metabolites were averaged, along with

a two-way ANOVA and Sidak multiple comparisons for both the embryo and larval
metabolism tests.

Metric 22: Reporting of Data High Data for the metabolism study for embryos and larva were presented.

Metric 23: Explanation of Unexpected Outcomes High There were no unexpected outcomes for the metabolism study.

Additional Comments: Metabolism study for zebrafish embryos accompanied by the larval metabolism study.

Overall Quality Determination Uninformative

Page 19 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 4290535 Table: 1 of 2

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Du, Z., Wang, G., Gao, S., Wang, Z. (2015). Aryl organophosphate flame retardants induced cardiotoxicity during zebrafish embryogenesis: by disturbing
expression of the transcriptional regulators. Aquatic Toxicology 16125-32.

Overall Duration: 0 - 4 days (0-96h); Exposure Duration: 0 - 4 days (0-96h)

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ AB Strain; Embryo
Cardiovascular

Tris(2-chloroethyl) phosphate (TCEP)

4290535

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1:
Metric 2:
Metric 3:

Test Substance Identity
Test Substance Source
Test Substance Purity

High
Low
High

toxicant identified by acronym and CAS number
Sigma"Aldrich was the supplier, not analytically verified
purity reported as 97%

Domain 2: Test Design

Metric 4:
Metric 5:

Metric 6:

Negative Controls
Negative Control Response

Randomized Allocation

High
Medium

Low

suitable solvent control was used

author reported strictly following OECD guidelines which, if valid, indicates that control

survival was >=90%

no mention of randomization reported

Domain 3: Exposure Characterization
Metric 7:

Metric 8:

Metric 9:

Metric 10:
Metric 11:

Metric 12:

Experimental System/Test Media
Preparation

Consistency of Exposure
Administration

Measurement of Test Substance
Concentration

Exposure Duration and Frequency
Number of Exposure Groups/
Spacing of Exposure Levels
Testing at or Below Solubility Limit

High

Medium

Low

High
Uninformative

High

daily renewal is adequate

not enough details provided to warrant a high rating
chemical concentrations were not measured
followed oecd guidelines for acute tests

It was just stated that seven concentrations were used per chemical. There was no infor-
mation on the test concentration used.

EC50 was well below solubility limit

Domain 4: Test Organism

Metric 13:
Metric 14:

Metric 15:

Test Organism Characteristics
Acclimatization and Pretreatment
Conditions

Number of Organisms and
Replicates per Group

High
High

Medium

well documented and met OEECD guidelines
reasonably well documented, seemed adequate

followed OECD guidelines for organism number

Domain 5: Outcome Assessment

Metric 16:
Metric 17:

Adequacy of Test Conditions
Outcome Assessment Methodology

High
High

enough documentation to verify adequacy
simple and consistent methods used

Continued on next page ...

Page 20 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 4290535 Table: 1 of 2

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Du, Z., Wang, G., Gao, S., Wang, Z. (2015). Aryl organophosphate flame retardants induced cardiotoxicity during zebrafish embryogenesis: by disturbing
expression of the transcriptional regulators. Aquatic Toxicology 16125-32.

Overall Duration: 0 - 4 days (0-96h); Exposure Duration: 0 - 4 days (0-96h)

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ AB Strain; Embryo
Cardiovascular

Tris(2-chloroethyl) phosphate (TCEP)

4290535

Domain

Metric

Rating

Comments

Metric 18:

Consistency of Outcome
Assessment

Medium

methods used to quantify pericardial edema were not well documented

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure

High
High

no variables were reported in the study
no unexpected outcomes were reported

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods

Metric 22: Reporting of Data

Metric 23: Explanation of Unexpected Outcomes

High
High
High

methods of calculating EC50s were reported
clear reporting of EC50s
no unexpected outcomes reported

Additional Comments: None

Overall Quality Determination

Uninformative

Page 21 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 4290535 Table: 2 of 2

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Du, Z., Wang, G., Gao, S., Wang, Z. (2015). Aryl organophosphate flame retardants induced cardiotoxicity during zebrafish embryogenesis: by disturbing
expression of the transcriptional regulators. Aquatic Toxicology 16125-32.

Overall Duration: 0 - 4 days (0-96h); Exposure Duration: 0 - 4 days (0-96h)

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ AB Strain; Embryo
Mortality

Tris(2-chloroethyl) phosphate (TCEP)

4290535

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1:
Metric 2:
Metric 3:

Test Substance Identity
Test Substance Source
Test Substance Purity

High
Low
High

toxicant identified by acronym and CAS number
Sigma"Aldrich was the supplier, not analytically verified
purity reported as 97%

Domain 2: Test Design

Metric 4:
Metric 5:

Metric 6:

Negative Controls
Negative Control Response

Randomized Allocation

High
Medium

Low

suitable solvent control was used

author reported strictly following OECD guidelines which, if valid, indicates that control

survival was >=90%

no mention of randomization reported

Domain 3: Exposure Characterization
Metric 7:

Metric 8:

Metric 9:

Metric 10:
Metric 11:

Metric 12:

Experimental System/Test Media
Preparation

Consistency of Exposure
Administration

Measurement of Test Substance
Concentration

Exposure Duration and Frequency
Number of Exposure Groups/
Spacing of Exposure Levels
Testing at or Below Solubility Limit

High

Medium

Low

High
Uninformative

High

daily renewal is adequate

not enough details provided to warrant a high rating
chemical concentrations were not measured
followed oecd guidelines for acute tests

It was just stated that seven concentrations were used per chemical. There was no infor-
mation on the test concentration used
LC50 was well below solubility limit

Domain 4: Test Organism

Metric 13:
Metric 14:

Metric 15:

Test Organism Characteristics
Acclimatization and Pretreatment
Conditions

Number of Organisms and
Replicates per Group

High
High

Medium

well documented and met OEECD guidelines
reasonably well documented, seemed adequate

followed OECD guidelines for organism number

Domain 5: Outcome Assessment

Metric 16:
Metric 17:
Metric 18:

Adequacy of Test Conditions
Outcome Assessment Methodology
Consistency of Outcome
Assessment.

High
High
High

enough documentation to verify adequacy
simple and consistent methods used
simple and consistent methods used

Continued on next page ...

Page 22 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 4290535 Table: 2 of 2

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Du, Z., Wang, G., Gao, S., Wang, Z. (2015). Aryl organophosphate flame retardants induced cardiotoxicity during zebrafish embryogenesis: by disturbing
expression of the transcriptional regulators. Aquatic Toxicology 16125-32.

Overall Duration: 0 - 4 days (0-96h); Exposure Duration: 0 - 4 days (0-96h)

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ AB Strain; Embryo
Mortality

Tris(2-chloroethyl) phosphate (TCEP)

4290535

Domain

Metric

Rating

Comments

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure

High
High

no variables were reported in the study
no unexpected outcomes were reported

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods

Metric 22: Reporting of Data

Metric 23: Explanation of Unexpected Outcomes

High
High
High

methods of calculating LC50s were reported
clear reporting of LC50s
no unexpected outcomes reported

Additional Comments: None

Overall Quality Determination

Uninformative



Page 23 of 170

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December 2023

Tris(2-chloroethyl) phosphate (TCEP) Environmental Hazard Evaluation HERO ID: 4180931 Table: 1 of 1

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Jarema, K. A., Hunter, D. L., Shaffer, R. M., Behl, M., Padilla, S. (2015). Acute and developmental behavioral effects of flame retardants and related
chemicals in zebrafish. Neurotoxicology and Teratology 52(Pt B):194-209.

Overall Duration: 4-10 days; Exposure Duration: 0 - 4 days (0-96h)

Aquatic (freshwater); Cell Culture Media; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact
uptake route)

Vertebrate; Fish; Danio rerio\ wild type; Larvae
Behavioral

Tris(2-chloroethyl) phosphate (TCEP)

4180931

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1: Test Substance Identity
Metric 2: Test Substance Source

Metric 3: Test Substance Purity

High Well documented in supplemental data and table 1. IUPAC name and structure provided.
Low NTP is listed as supplier, Sigma-Aldrich as the manufacturer but no analytical verifica-
tion was reported

High Information is in supplemental data Table 1, purity of 99% reported.

Domain 2: Test Design

Metric 4: Negative Controls
Metric 5: Negative Control Response
Metric 6: Randomized Allocation

High DMSO vehicle control was used as negative control, and chlorpyrifos or heptachlor were
used as positive controls.

Low No report on survival or malformation rate for controls, no comparison between clean
and solvent controls were reported.

Medium Dose groups were randomized. "The location of each chemical concentration was ran-
domized on the stock plate (96 well glass plate which mirrored the dosing on the ex-
perimental plate containing the embryos/larvae), and therefore the dose groups on the
experimental plate were also randomized."

Domain 3: Exposure Characterization

Metric 7:

Experimental System/Test Media

High

Experimental system was explained in detail.

Preparation

Metric 8:

Consistency of Exposure

High

Method for administering the test substance was reported in detail, and was consistent

Administration

across study groups.

Metric 9:

Measurement of Test Substance

Low

Test substance concentrations were not analytically verified. Wells were covered and

Concentration

sealed with Parafilm to minimize evaporation.

Metric 10:

Exposure Duration and Frequency

High

durations were based on other studies

Metric 11:

Number of Exposure Groups/

High

Five concentrations were tested, covering an adequate range for a response. A range

Spacing of Exposure Levels

finding study was conducted to set the highest dose that would not cause any acute ef-

fects or lethality that would confound the behavioral assessment.

Metric 12:

Testing at or Below Solubility Limit

Medium

No indication given for how solvent concentrations were chosen.

Domain 4: Test Organism

Metric 13: Test Organism Characteristics

High "Wild type adult zebrafish (Danio rerio), undefined, outbred stock originally obtained
from Aquatic Research Organisms, Hampton, NH, 03842 and EkkWill Waterlife Re-
sources, Ruskin, FL 33575 were" used for egg collection. Housing conditions for adult
zebrafish and procedure for collecting eggs was described in detail.

Continued on next page ...

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Environmental Hazard Evaluation

HERO ID: 4180931 Table: 1 of 1

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Overall Duration: 4-10 days; Exposure Duration: 0 - 4 days (0-96h)

Aquatic (freshwater); Cell Culture Media; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact
uptake route)

Vertebrate; Fish; Danio rerio\ wild type; Larvae
Behavioral

Tris(2-chloroethyl) phosphate (TCEP)

4180931

Domain

Metric

Rating

Comments

Metric 14:
Metric 15:

Acclimatization and Pretreatment
Conditions

Number of Organisms and
Replicates per Group

High
Medium

Testing paradigm began with a 20 minute acclimation phase; all control and test organ-
isms were subjected to the same pretreatment conditions.

The number tested at each concentration were reported - 24 larvae per dose.

Domain 5: Outcome Assessment

Metric 16: Adequacy of Test Conditions

Metric 17: Outcome Assessment Methodology
Metric 18: Consistency of Outcome
Assessment.

Medium Conditions seemed adequate for embryo development, small quantities are difficult to

assess.

High Well documented assessment procedure.

High No deviations in assessment procedures were reported.

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure

High No confounding variables were reported.

Medium No unrelated outcomes reported downgraded because only normal larvae were assessed.

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods High

Metric 22: Reporting of Data High

Metric 23: Explanation of Unexpected Outcomes Medium

Well documented analysis methods.

All behavioral data was presented in a reasonable manner.

Downgraded because they only assessed normal larvae and none during the acclimation
phase.

Additional Comments: None

Overall Quality Determination

High

Page 25 of 170

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December 2023

Environmental Hazard Evaluation

HERO ID: 7274629 Table: 1 of 3

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Lee, J. S., Morita, Y., Kawai, Y. K., Covaci, A., Kubota, A. (2020). Developmental circulatory failure caused by metabolites of organophosphorus flame

retardants in zebrafish, Danio rerio. Chemosphere 246125738.

Overall Duration: 0 - 4 days (0-96h); Exposure Duration: 0 - 4 days (0-96h)

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ Embryo
Cardiovascular

Tris(2-chloroethyl) phosphate (TCEP)

7274629

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1: Test Substance Identity
Metric 2: Test Substance Source

Metric 3: Test Substance Purity

High Chemical was identified by name and structure presented in Figure 1.

High Test substance source was identified and test substance purity was measured by MS and

NMR techniques.

High Purity was measured at >98%

Domain 2: Test Design

Metric 4: Negative Controls
Metric 5: Negative Control Response

Metric 6: Randomized Allocation

High DMSO controls were run concurrently.

High The biological response of the negative control group was reported and reasonable for
assessed outcomes

Low Researchers did not report how organisms were allocated to study groups

Domain 3: Exposure Characterization

Metric 7:

Experimental System/Test Media

High

The experimental system and methods for preparation of test media were described in



Preparation



adequate detail

Metric 8:

Consistency of Exposure

High

Details of exposure administration were reported and exposures were administered



Administration



consistently across study groups.

Metric 9:

Measurement of Test Substance

Low

Exposure concentrations were not measured or measurements were not reported.



Concentration





Metric 10:

Exposure Duration and Frequency

High

The duration of exposure was reported and suitable for the study type

Metric 11:

Number of Exposure Groups/

High

Concentration spacing was adequate for the developmental experiment.



Spacing of Exposure Levels





Metric 12:

Testing at or Below Solubility Limit

High

Exposure concentrations were below reported water solubility limits and a solvent was

employed to further aid solubility

Domain 4: Test Organism

Metric 13: Test Organism Characteristics	Medium

Metric 14: Acclimatization and Pretreatment	Low

Conditions

Metric 15: Number of Organisms and Medium
	Replicates per Group	

Test organism source was not stated, otherwise the organisms were described ade-
quately.

The study did not report whether test organisms were acclimatized and/or whether pre-
treatment conditions were the same for control and exposed groups
The numbers of test organisms and replicates were reported and sufficient to character-
ize toxicological effects. 10 organisms per dish with 3 replicates per treatment level.

Domain 5: Outcome Assessment

Continued on next page ...

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 7274629 Table: 1 of 3

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Lee, J. S., Morita, Y., Kawai, Y. K., Covaci, A., Kubota, A. (2020). Developmental circulatory failure caused by metabolites of organophosphorus flame

retardants in zebrafish, Danio rerio. Chemosphere 246125738.

Overall Duration: 0 - 4 days (0-96h); Exposure Duration: 0 - 4 days (0-96h)

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ Embryo
Cardiovascular

Tris(2-chloroethyl) phosphate (TCEP)

7274629

Domain

Metric

Rating

Comments

Metric 16: Adequacy of Test Conditions Medium

Metric 17: Outcome Assessment Methodology High

Metric 18: Consistency of Outcome High

Assessment

They state the embryos were reared in breeding water. There are uncertainties on
whether this is the same water the adults were bred in (the assumption would be yes)
and if the temperature is the same in the static test dishes as it is in the flow through
tanks.

The outcome assessment methodology addressed or reported the intended outcome of
interest. Reduction in blood flow was measured by degree of circulation of red blood
cells through a certain region of a trunk vessel.

Embryos were exposed at 72 hpf and assessed for reduction in blood flow at 96h using a
0-2 grading system under an inverted microscope.

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test High

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure Medium

There were no reported differences among the study groups in environmental conditions
or other factors that could influence the outcome assessment

There was no information in the study to suggest differences among groups in animal
attrition or health outcomes unrelated to exposure

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods High

Metric 22: Reporting of Data High

Metric 23: Explanation of Unexpected Outcomes High

Statistical methods were adequately described

Results on Blood Flow are presented in Supplemental Data Figure S2

There were no unexpected outcomes.

Additional Comments:

The cardiovascular outcome was chosen for this because it pertains to circulation of red blood cells and cardiac function.Data for this form are contained
in Supplemental Figure S2. TCEP metabolite BCEP was also studied in this test.

Overall Quality Determination

High

Page 27 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 7274629 Table: 2 of 3

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Lee, J. S., Morita, Y., Kawai, Y. K., Covaci, A., Kubota, A. (2020). Developmental circulatory failure caused by metabolites of organophosphorus flame

retardants in zebrafish, Danio rerio. Chemosphere 246125738.

Overall Duration: 0 - 4 days (0-96h); Exposure Duration: 0 - 4 days (0-96h)

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ Embryo
Development/Growth
Tris(2-chloroethyl) phosphate (TCEP)

7274629

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

Chemical Structure was presented in Figure 1.

Metric 2:

Test Substance Source

High

Test substance source was identified and test substance purity was measured by MS and

NMR techniques.

Metric 3:

Test Substance Purity

High

Purity was measured at >98%

Domain 2: Test Design

Metric 4:

Negative Controls

High

DMSO controls were run concurrently.

Metric 5:

Negative Control Response

High

The biological response of the negative control group was reported and reasonable for

assessed outcomes

Metric 6:

Randomized Allocation

Low

Random allocation was not detailed.

Domain 3: Exposure Characterization

Metric 7:

Experimental System/Test Media

High

The experimental system and methods for preparation of test media were described in

Preparation

adequate detail

Metric 8:

Consistency of Exposure

High

Details of exposure administration were reported and exposures were administered

Administration

consistently across study groups.

Metric 9:

Measurement of Test Substance

Low

Exposure concentrations were not measured or measurements were not reported.

Concentration

Metric 10:

Exposure Duration and Frequency

High

The duration of exposure was reported and suitable for the study type

Metric 11:

Number of Exposure Groups/

High

concentration spacing was adequate for the developmental experiment.

Spacing of Exposure Levels

Metric 12:

Testing at or Below Solubility Limit

High

Exposure concentrations were below reported water solubility limits and a solvent was

employed to further aid solubility.

Domain 4: Test Organism

Metric 13:

Test Organism Characteristics

Medium

Test organism source was not stated, otherwise the organisms were described ade-

quately.

Metric 14:

Acclimatization and Pretreatment

Low

The study did not report whether test organisms were acclimatized and/or whether pre-

Conditions

treatment conditions were the same for control and exposed groups

Metric 15:

Number of Organisms and

Medium

The numbers of test organisms and replicates were reported and sufficient to character-

Replicates per Group

ize toxicological effects. 10 organisms per dish with 3 replicates per treatment level.

Domain 5: Outcome Assessment

Continued on next page ...

Page 28 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 7274629 Table: 2 of 3

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Lee, J. S., Morita, Y., Kawai, Y. K., Covaci, A., Kubota, A. (2020). Developmental circulatory failure caused by metabolites of organophosphorus flame

retardants in zebrafish, Danio rerio. Chemosphere 246125738.

Overall Duration: 0 - 4 days (0-96h); Exposure Duration: 0 - 4 days (0-96h)

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ Embryo
Development/Growth
Tris(2-chloroethyl) phosphate (TCEP)

7274629

Domain

Metric

Rating

Comments

Metric 16: Adequacy of Test Conditions Medium

Metric 17: Outcome Assessment Methodology High

Metric 18: Consistency of Outcome High

Assessment

The outcome assessment methodology addressed or reported the intended outcome of
interest. Pericardial edema was assessed at different treatment levels.

Embryos were exposed at 72 hpf and assessed for pericardial edema at 96h using a 0-2
grading system under an inverted microscope.

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test High

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure Medium

There were no reported differences among the study groups in environmental conditions
or other factors that could influence the outcome assessment

There was no information in the study to suggest differences among groups in animal
attrition or health outcomes unrelated to exposure

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods
Metric 22: Reporting of Data

Metric 23: Explanation of Unexpected Outcomes

High Statistical methods were adequately described

High Data for exposure-related findings were presented for each treatment and control group

and were adequate to determine values for the endpoint of interest
High There were no unexpected outcomes.

Additional Comments:

Development/growth was selected because pericardial edema was being assessed as a morphological abnormality seen during an early life stagePE data
are presented in Figure 4, Page 4/7. TCEP metabolite BCEP was also tested in this study.

Overall Quality Determination

High

Page 29 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 7274629 Table: 3 of 3

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Lee, J. S., Morita, Y., Kawai, Y. K., Covaci, A., Kubota, A. (2020). Developmental circulatory failure caused by metabolites of organophosphorus flame

retardants in zebrafish, Danio rerio. Chemosphere 246125738.

Overall Duration: 0 - 4 days (0-96h); Exposure Duration: 0 - 4 days (0-96h)

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ Embryo
Mortality

Tris(2-chloroethyl) phosphate (TCEP)

7274629

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

Chemical Structure was presented in Figure 1.

Metric 2:

Test Substance Source

High

Test substance source was identified and test substance purity was measured by MS and

NMR techniques.

Metric 3:

Test Substance Purity

High

Purity was measured at >98%

Domain 2: Test Design

Metric 4:

Negative Controls

High

DMSO controls were run concurrently.

Metric 5:

Negative Control Response

High

The biological response (mortality) of the negative control group was reported and rea-

sonable for assessed outcomes

Metric 6:

Randomized Allocation

Low

Random allocation was not detailed.

Domain 3: Exposure Characterization

Metric 7:

Experimental System/Test Media

High

The experimental system and methods for preparation of test media were described in

Preparation

adequate detail

Metric 8:

Consistency of Exposure

High

Details of exposure administration were reported and exposures were administered

Administration

consistently across study groups.

Metric 9:

Measurement of Test Substance

Low

Exposure concentrations were not measured or measurements were not reported.

Concentration

Metric 10:

Exposure Duration and Frequency

High

The duration of exposure was reported and suitable for the study type

Metric 11:

Number of Exposure Groups/

High

concentration spacing was adequate for the developmental experiment.

Spacing of Exposure Levels

Metric 12:

Testing at or Below Solubility Limit

High

Exposure concentrations were below reported water solubility limits and a solvent was

employed to further aid solubility.

Domain 4: Test Organism

Metric 13:

Test Organism Characteristics

Medium

Test organism source was not stated, otherwise the organisms were described ade-

quately.

Metric 14:

Acclimatization and Pretreatment

Low

The study did not report whether test organisms were acclimatized and/or whether pre-

Conditions

treatment conditions were the same for control and exposed groups

Metric 15:

Number of Organisms and

Medium

The numbers of test organisms and replicates were reported and sufficient to character-

Replicates per Group

ize toxicological effects. 10 organisms per dish with 3 replicates per treatment level.

Domain 5: Outcome Assessment

Continued on next page ...

Page 30 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 7274629 Table: 3 of 3

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Lee, J. S., Morita, Y., Kawai, Y. K., Covaci, A., Kubota, A. (2020). Developmental circulatory failure caused by metabolites of organophosphorus flame

retardants in zebrafish, Danio rerio. Chemosphere 246125738.

Overall Duration: 0 - 4 days (0-96h); Exposure Duration: 0 - 4 days (0-96h)

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ Embryo
Mortality

Tris(2-chloroethyl) phosphate (TCEP)

7274629

Domain

Metric

Rating

Comments

Metric 16: Adequacy of Test Conditions Medium

Metric 17: Outcome Assessment Methodology High

Metric 18: Consistency of Outcome Medium

Assessment

The outcome assessment methodology addressed or reported the intended outcome of
interest. Pericardial edema was assessed at different treatment levels.

Embryos were exposed at 72 hpf and assessed Mortality at 96h. Specific criteria for
mortality (ie, heart beat, response to stimuli, etc) was not reported.

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test High

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure Medium

There were no reported differences among the study groups in environmental conditions
or other factors that could influence the outcome assessment

There was no information in the study to suggest differences among groups in animal
attrition or health outcomes unrelated to exposure

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods
Metric 22: Reporting of Data

Metric 23: Explanation of Unexpected Outcomes

High Statistical methods were adequately described

High Data for exposure-related findings were presented for each treatment and control group

and were adequate to determine values for the endpoint of interest
High There were no unexpected outcomes.

Additional Comments:

This form represents the mortality observations reported in Fig 4 and Fig S2. TCEP exposures resulted in no mortality observations in the recorded data,
however, mortality was reported as being recorded for all compounds in this study within the materials and methods.

Overall Quality Determination

High

Page 31 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 2953504 Table: 1 of 3

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Noyes, P. D., Haggard, D. E., Gonnerman, G. D., Tanguay, R. L. (2015). Advanced morphological - behavioral test platform reveals neurodevelopmental
defects in embryonic zebrafish exposed to comprehensive suite of halogenated and organophosphate flame retardants. Toxicological Sciences 145(1):177-
195.

Overall Duration: 0 - 4 days (0-96h); Exposure Duration: 0 - 4 days (0-96h)

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ wild type (Tropical 5D); Embryo
Mortality

Tris(2-chloroethyl) phosphate (TCEP)

2953504

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

The chemical is referred to by name, structure, and CASRN.

Metric 2:

Test Substance Source

Low

Source was reported but the authors did not perform analytical verification.

Metric 3:

Test Substance Purity

High

Purity is >98% and reported in Table 3.

Domain 2: Test Design

Metric 4: Negative Controls
Metric 5: Negative Control Response
Metric 6: Randomized Allocation

High A solvent control (DMSO at 0.64% ) was used.

High All control responses are reported in the supplemental data PDF.

Medium Although not specifically reported as "random" the process of chorion removal and
distribution into well plates was automated and is detailed in: "Mandrell, David, Lisa
Truong, Caleb Jephson, Mushfiqur R. Sarker, Aaron Moore, Christopher Lang, Michael
T. Simonich, and Robert L. Tanguay. "Automated zebrafish chorion removal and single
embryo placement: optimizing throughput of zebrafish developmental toxicity screens."
Journal of laboratory automation 17, no. 1 (2012): 66-74."

Domain 3: Exposure Characterization

Metric 7:

Experimental System/Test Media

Medium

Preparation

Metric 8:

Consistency of Exposure

High

Administration

Metric 9:

Measurement of Test Substance

Low

Concentration

Metric 10:

Exposure Duration and Frequency

High

Metric 11:

Number of Exposure Groups/

High

Spacing of Exposure Levels

Metric 12:

Testing at or Below Solubility Limit

Medium

The exposures were static non-renewal from 6 to 120 hpf. Renewal of compounds
would have been preferred.

Exposures were conducted consistently among treatments and control.

No verification was performed, concentrations are reported as nominal.

Exposure was initiated at 6 hpf and continued till 120 hpf. The assessments at 24 and
120 hpf are appropriate to capture embryo and larval periods.

Concentrations were: 64, 6.4, 0.64, 0.064, 0.0064, and 0 uM and represent a broad range
of exposure concentrations.

The highest concentration (64 uM) is above the solubility limit. The remaining four con-
centrations are below solubility for TBBPA and TCEP. The highest two concentrations
(6.4 and 64 uM) are above TPP solubility limit (solubility from final scopes for TBBPA,
TPP, and TCEP are 4.1, 1.9 and 7.8 mg/L)

Domain 4: Test Organism

Metric 13: Test Organism Characteristics

High The strain and source for broodfish was reported.

Continued on next page ...

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 2953504 Table: 1 of 3

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Overall Duration: 0 - 4 days (0-96h); Exposure Duration: 0 - 4 days (0-96h)

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ wild type (Tropical 5D); Embryo
Mortality

Tris(2-chloroethyl) phosphate (TCEP)

2953504

Domain

Metric

Rating

Comments

Metric 14:
Metric 15:

Acclimatization and Pretreatment
Conditions

Number of Organisms and
Replicates per Group

High
Medium

Water quality parameters were well described and accordance with protocols under
Oregon State University"s Institutional Animal Care and Use Committee.

All sample size numbers for treatment and control groups are listed in the first two pages
of the supplemental data PDF for this work.

Domain 5: Outcome Assessment

Metric 16:

Adequacy of Test Conditions

Medium

Photoperiod and temperature were reported, while other water quality conditions were
not.

Mortality assessment was described in the section titled "Developmental malformation
evaluations" but authors did not report specific criteria for death (ie, movement, heart-
beat, color, etc.).

Assessment was consistent among treatments and control.

Metric 17:
Metric 18:

Outcome Assessment Methodology

Consistency of Outcome
Assessment

Medium
High

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure

High
Medium

Nothing was reported to indicate differences in study groups based on environmental
factors.

No information in the study to indicate that differences were from animal attrition or

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods

Metric 22: Reporting of Data

Metric 23: Explanation of Unexpected Outcomes

High "Data collection was undertaken using a custom barcoding and tracking system (Ze-
brafish Acquisition and Analysis Program) to facilitate reliable management of the large
amounts of data collected. Statistical analyses were performed using R code with test-
ing methodologies used by Truong et al. (2014) to evaluate developmental toxicity of
chemicals under the ToxCast program(RCoreTeam, 2014; Truong et al., 2014). Briefly,
a binomial testwas performed that calculated lowest effect levels (LELs) foreach end-
point to identify incidences that exceeded a significantthreshold above controls. This test
was preferable to a logisticregression as it accounted for the observed nonmonotonicity
offlame retardant toxicity."

High All counts of data per treatment and control group are represented in the supplemental
data for each compound and time point (24 and 120 hpf).

High No unexpected outcomes were reported by the authors.

Additional Comments: This form is for Mortality assessment at 24 hpf for TCEP. The specific data and statistical significance for each compound are located within the supple-
mental PDF.

Continued on next page ...

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 2953504 Table: 1 of 3

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Noyes, P. D., Haggard, D. E., Gonnerman, G. D., Tanguay, R. L. (2015). Advanced morphological - behavioral test platform reveals neurodevelopmental
defects in embryonic zebrafish exposed to comprehensive suite of halogenated and organophosphate flame retardants. Toxicological Sciences 145(1):177-
195.

Overall Duration: 0 - 4 days (0-96h); Exposure Duration: 0 - 4 days (0-96h)

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ wild type (Tropical 5D); Embryo
Mortality

Tris(2-chloroethyl) phosphate (TCEP)

2953504

Domain Metric

Rating

Comments

Overall Quality Determination

High





Page 34 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

PUBLIC RELEASE DRAFT - DO NOT CITE OR QUOTE
December 2023

Environmental Hazard Evaluation

HERO ID: 2953504 Table: 2 of 3

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Overall Duration: 0 - 4 days (0-96h); Exposure Duration: 0 - 4 days (0-96h)

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ wild type (Tropical 5D); Embryo
Behavioral

Tris(2-chloroethyl) phosphate (TCEP)

2953504

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

The chemical is referred to by name, structure, and CASRN.

Metric 2:

Test Substance Source

Low

Source was reported but the authors did not perform analytical verification.

Metric 3:

Test Substance Purity

High

Purity is >98% and reported in Table 3.

Domain 2: Test Design

Metric 4: Negative Controls
Metric 5: Negative Control Response
Metric 6: Randomized Allocation

High A solvent control (DMSO at 0.64% ) was used.

High All control responses are reported in the supplemental data PDF.

Domain 3: Exposure Characterization

Metric 7:

Experimental System/Test Media

Medium

Preparation

Metric 8:

Consistency of Exposure

High

Administration

Metric 9:

Measurement of Test Substance

Low

Concentration

Metric 10:

Exposure Duration and Frequency

High

Metric 11:

Number of Exposure Groups/

High

Spacing of Exposure Levels

Metric 12:

Testing at or Below Solubility Limit

Medium

would have been preferred.

Exposures were conducted consistently among treatments and control.

No verification was performed, concentrations are reported as nominal.

Exposure was initiated at 6 hpf and continued till 120 hpf. The assessments at 24 and
120 hpf are appropriate to capture embryo and larval periods.

Concentrations were: 64, 6.4, 0.64, 0.064, 0.0064, and 0 uM and represent a broad range
of exposure concentrations.

Domain 4: Test Organism

Metric 13: Test Organism Characteristics High

Metric 14: Acclimatization and Pretreatment High

Conditions

The strain and source for broodfish was reported.

Water quality parameters were well described and accordance with protocols under
Oregon State University"s Institutional Animal Care and Use Committee.

Continued on next page ...

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 2953504 Table: 2 of 3

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Overall Duration: 0 - 4 days (0-96h); Exposure Duration: 0 - 4 days (0-96h)

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ wild type (Tropical 5D); Embryo
Behavioral

Tris(2-chloroethyl) phosphate (TCEP)

2953504

Domain

Metric

Rating

Comments

Metric 15:

Number of Organisms and
Replicates per Group

Medium

All sample size numbers for treatment and control groups are listed in the first two pages
of the supplemental data PDF for this work.

Domain 5: Outcome Assessment

Metric 16:

Adequacy of Test Conditions

Medium

Photoperiod and temperature were reported, while other water quality conditions were
not.

Movement assays for embryos (24 hpf) and larvae (120 hpf) were described in detail on
page 7 of 19.

Assessment was consistent among treatments and control.

Metric 17:
Metric 18:

Outcome Assessment Methodology

Consistency of Outcome
Assessment.

Medium
High

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure

High Nothing was reported to indicate differences in study groups based on environmental
factors.

Medium No information in the study to indicate that differences were from animal attrition or
health outcomes.

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods

Metric 22: Reporting of Data

Metric 23: Explanation of Unexpected Outcomes

High Embryo movement: "Specifically, overall patterns of activity within each cycle inter-
val (ie, baseline, excitation, refractory) were compared with those in vehicle controls
by (1) estimating the 50% peak difference from controls in either direction and (2)
performing a Kolmogorov"Smirnov test that compared the empirical cumulative dis-
tribution function between chemical treatments and controls. A Bonferroni-corrected
p-value threshold of .01 (0.05/5 treatments".01) was used to determine statistical signif-
icance. "Larval Movement: "As larval activity did not meet parametric assumptions of
normality, Kruskal"Wallis analyses of variance and Dunn"s multiple comparison post
tests were used to compare median locomotor activity per minute in treatment versus
controls in each of the 5-min light/dark phases."

High All raw data across time for embryo and larval movement is reported in the supplemen-
tal notes (24 and 120 hpf).

High No unexpected outcomes were reported by the authors.

Additional Comments: This form is for embryo movement assessment at 24 hpf for TCEP.

Overall Quality Determination

High

Page 36 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 2953504 Table: 3 of 3

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Noyes, P. D., Haggard, D. E., Gonnerman, G. D., Tanguay, R. L. (2015). Advanced morphological - behavioral test platform reveals neurodevelopmental
defects in embryonic zebrafish exposed to comprehensive suite of halogenated and organophosphate flame retardants. Toxicological Sciences 145(1):177-
195.

Overall Duration: 0 - 4 days (0-96h); Exposure Duration: 0 - 4 days (0-96h)

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ wild type (Tropical 5D); Embryo
Development/Growth
Tris(2-chloroethyl) phosphate (TCEP)

2953504

Domain

Metric

Rating

Comments

Domain 1: Test Substance







Metric 1:

Test Substance Identity

High

The chemical is referred to by name, structure, and CASRN.

Metric 2:

Test Substance Source

Low

Source was reported but the authors did not perform analytical verification.

Metric 3:

Test Substance Purity

High

Purity is >98% and reported in Table 3.

Domain 2: Test Design

Metric 4: Negative Controls
Metric 5: Negative Control Response
Metric 6: Randomized Allocation

High A solvent control (DMSO at 0.64% ) was used.

High All control responses are reported in the supplemental data PDF.

Medium Although not specifically reported as "random" the process of chorion removal and
distribution into well plates was automated and is detailed in: "Mandrell, David, Lisa
Truong, Caleb Jephson, Mushfiqur R. Sarker, Aaron Moore, Christopher Lang, Michael
T. Simonich, and Robert L. Tanguay. "Automated zebrafish chorion removal and single
embryo placement: optimizing throughput of zebrafish developmental toxicity screens."
Journal of laboratory automation 17, no. 1 (2012): 66-74."

Domain 3: Exposure Characterization

Metric 7:

Experimental System/Test Media

Medium



Preparation



Metric 8:

Consistency of Exposure

High



Administration



Metric 9:

Measurement of Test Substance

Low



Concentration



Metric 10:

Exposure Duration and Frequency

High

Metric 11: Number of Exposure Groups/
Spacing of Exposure Levels

High

would have been preferred.

Exposures were conducted consistently among treatments and control.

No verification was performed, concentrations are reported as nominal.

Exposure was initiated at 6 hpf and continued till 120 hpf. The assessments at 24 and
120 hpf are appropriate to capture embryo and larval periods. 24 hour developmental
assessments included: delays in developmental progression, notochord deformities, and
altered spontaneous movements. 120 hour developmental assessments included: 17 de-
velopmental malformations, including yolk sac edema (YSE) and pericardial edema
(PE); body axis (AXIS), trunk length (TRUN), caudal fin (CFIN), pectoral fin (PFIN),
pigmentation (PIG), and somite (SOMI) deformities; eye (EYE), snout (SNOU), jaw
(JAW), and otolith (OTIC) malformations; gross brain development (BRAIN); noto-
chord (NC) and circulatory (CIRC) deformities; swim bladder presence and inflation
(SWIM); and touch responses (TR).

Concentrations were: 64, 6.4, 0.64, 0.064, 0.0064, and 0 uM and represent a broad range
of exposure concentrations.

Continued on next page ...

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 2953504 Table: 3 of 3

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Overall Duration: 0 - 4 days (0-96h); Exposure Duration: 0 - 4 days (0-96h)

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ wild type (Tropical 5D); Embryo
Development/Growth
Tris(2-chloroethyl) phosphate (TCEP)

2953504

Domain

Metric

Rating

Comments

Metric 12:

Testing at or Below Solubility Limit

Medium

Domain 4: Test Organism

Metric 13:
Metric 14:

Metric 15:

Test Organism Characteristics
Acclimatization and Pretreatment
Conditions

Number of Organisms and
Replicates per Group

High
High

Medium

The strain and source for broodfish was reported.

Water quality parameters were well described and accordance with protocols under
Oregon State University"s Institutional Animal Care and Use Committee.

All sample size numbers for treatment and control groups are listed in the first two pages
of the supplemental data PDF for this work.

Domain 5: Outcome Assessment

Metric 16:

Adequacy of Test Conditions

Medium

Photoperiod and temperature were reported, while other water quality conditions were
not.

Developmental assessment was described in the section titled "Developmental malfor-
mation evaluations". The specifc analysis is detailed in another publication that authors
referenced as HERO ID 8591199

Assessment was consistent among treatments and control.

Metric 17:
Metric 18:

Outcome Assessment Methodology

Consistency of Outcome
Assessment

Medium
High

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure

High
Medium

Nothing was reported to indicate differences in study groups based on environmental
factors.

No information in the study to indicate that differences were from animal attrition or
health outcomes.

Domain 7: Data Presentation and Analysis

Continued on next page .

Page 38 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 2953504 Table: 3 of 3

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Noyes, P. D., Haggard, D. E., Gonnerman, G. D., Tanguay, R. L. (2015). Advanced morphological - behavioral test platform reveals neurodevelopmental
defects in embryonic zebrafish exposed to comprehensive suite of halogenated and organophosphate flame retardants. Toxicological Sciences 145(1):177-
195.

Overall Duration: 0 - 4 days (0-96h); Exposure Duration: 0 - 4 days (0-96h)

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ wild type (Tropical 5D); Embryo
Development/Growth
Tris(2-chloroethyl) phosphate (TCEP)

2953504

Domain

Metric

Rating

Comments

Metric 21:

Statistical Methods

High

"Data collection was undertaken using a custom barcoding and tracking system (Ze-
brafish Acquisition and Analysis Program) to facilitate reliable management of the large
amounts of data collected. Statistical analyses were performed using R code with test-
ing methodologies used by Truong et al. (2014) to evaluate developmental toxicity of
chemicals under the ToxCast program(RCoreTeam, 2014; Truong et al., 2014). Briefly,
a binomial testwas performed that calculated lowest effect levels (LELs) foreach end-
point to identify incidences that exceeded a significantthreshold above controls. This test
was preferable to a logisticregression as it accounted for the observed nonmonotonicity
offlame retardant toxicity."This reference is HERO ID 8591199

Metric 22:

Reporting of Data

High

All counts of data per treatment and control group are represented in the supplemental
data for each compound and time point (24 and 120 hpf).

Metric 23:

Explanation of Unexpected Outcomes

High

No unexpected outcomes were reported by the authors.

Additional Comments: This form is for developmental assessment at 24 hpf for TCEP. The specific data and statistical significance for each compound are located within the
	supplemental PDF.	

Overall Quality Determination

High

Page 39 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5166352 Table: 1 of 1

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Wang, G. W., Chen, H. Y., Du, Z. K., Li, J. H., Wang, Z. Y., Gao, S. X. (2017). In vivo metabolism of organophosphate flame retardants and distribution

of their main metabolites in adult zebraflsh. Science of the Total Environment 590:50-59.

Overall Duration: 0 - 4 days (0-96h); Exposure Duration: 0 - 4 days (0-96h)

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ Adult
Mortality

Tris(2-chloroethyl) phosphate (TCEP)

5166352

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1: Test Substance Identity
Metric 2: Test Substance Source
Metric 3: Test Substance Purity

High
Low
High

Chemical was identified by name, CAS# and structure. Details provided in Table SI.
The test substance identity was not analytically verified by the performing laboratory.
Chemical purity reported as >97% .

Domain 2: Test Design

Metric 4: Negative Controls

Metric 5: Negative Control Response
Metric 6: Randomized Allocation

Low

Low
Low

The use of a control is implied in order to calculate 96h LC 50 according to OECD 203
protocol.

The biological response of the negative control groups was not reported.

Researchers did not report how organisms were allocated to study groups.

Domain 3: Exposure Characterization

Metric 7:

Experimental System/Test Media

Low

The study provided no details on exposure media preparation for the mortality experi-

Preparation

ment.

Metric 8:

Consistency of Exposure

Low

The study provided no details on exposure administration for the mortality experiment.

Administration

Metric 9:

Measurement of Test Substance

Low

Exposure concentrations were not measured or measurements were not reported.

Concentration

Metric 10:

Exposure Duration and Frequency

High

The duration of exposure was reported (96 h) and appropriate for the study type.

Metric 11:

Number of Exposure Groups/

Uninformative

No information is provided on the number of exposure groups and spacing of exposure

Spacing of Exposure Levels

levels used to determine 96h LC 50.

Metric 12:

Testing at or Below Solubility Limit

Low

Reporting omissions prevented determination of whether exposure concentrations ex-

ceeded the water solubility limit.

Domain 4: Test Organism

Metric 13: Test Organism Characteristics

Metric 14: Acclimatization and Pretreatment

Conditions
Metric 15: Number of Organisms and

Replicates per Group

Medium The test organisms were obtained from a reliable source but specifics such as average

weight were not reported.

Low The study did not report whether test organisms were acclimatized.

Low The number of test organisms and/or replicates was not reported.

Domain 5: Outcome Assessment

Metric 16:

Adequacy of Test Conditions

Low

Environmental conditions were not sufficiently reported to evaluate if adequate and
whether differences occurred between control and exposed populations.

Continued on next page .

Page 40 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5166352 Table: 1 of 1

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Wang, G. W., Chen, H. Y., Du, Z. K., Li, J. H., Wang, Z. Y., Gao, S. X. (2017). In vivo metabolism of organophosphate flame retardants and distribution

of their main metabolites in adult zebrafish. Science of the Total Environment 590:50-59.

Overall Duration: 0 - 4 days (0-96h); Exposure Duration: 0 - 4 days (0-96h)

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ Adult
Mortality

Tris(2-chloroethyl) phosphate (TCEP)

5166352

Domain

Metric

Rating

Comments

Metric 17:
Metric 18:

Outcome Assessment Methodology

Consistency of Outcome
Assessment

Low
Low

The outcome assessment methodology was not clearly reported. It was stated that the
experiment was conducted according to OECD 203 but no other details were provided.
Details regarding the execution of the study protocol for outcome assessment were not
reported.

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure

Low
Medium

The study did not provide enough information to allow a comparison of environmental
conditions.

There was no information in the study to suggest differences among groups in animal
attrition or health outcomes unrelated to exposure (e.g., infection) that could influence
the outcome assessment.

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods
Metric 22: Reporting of Data

Metric 23: Explanation of Unexpected Outcomes

Low
Low

Low

Statistical analysis was performed but not described adequately.

Data for exposure-related findings were not shown for each treatment and control group,
but results were summarized in the text as an LC50 value.

The study did not report any measures of variability.

Additional Comments:

LC 50 values were determined according to OECD 203 but no details (regarding test media preparation, number of exposure groups, control response,
number of organisms and replicates, mortality data for each treatment group, environmental conditions, etc.) were provided. The main focus of the paper
was to study in vivo metabolism of TCEP and the LC50 values were used to set up the exposure groups for the main experiment.

Overall Quality Determination

Uninformative

Page 41 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 3479540 Table: 1 of 2

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Behl, M., Hsieh, J. H., Shafer, T. J., Mundy, W. R., Rice, J. R., Boyd, W. A., Freedman, J. H., Hunter, E. S., Jarema, K. A., Padilla, S., Tice, R. R. (2015).
Use of alternative assays to identify and prioritize organophosphorus flame retardants for potential developmental and neurotoxicity. Neurotoxicology and
Teratology 52(PtB):181-193.

Overall Duration: 4 - 10 days; Exposure Duration: 4 -10 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ Embryo
Development/Growth
Tris(2-chloroethyl) phosphate (TCEP)

3479540

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1: Test Substance Identity
Metric 2: Test Substance Source

Metric 3: Test Substance Purity

High Test substance was identified by name and CAS #

Low Test substance source was reported as Sigma-Aldrich, but it did not appear to be ana-

lyzed by the performing laboratory
High Test substance purity was 98.95%

Domain 2: Test Design

Metric 4: Negative Controls
Metric 5: Negative Control Response

Metric 6: Randomized Allocation

High All treated groups were compared to a DMSO vehicle controls.

Low The biological response of the negative control groups was not reported. The biological

response of the positive TOCP control was reported though.

Low Researchers did not report how organisms were allocated to study groups.

Domain 3: Exposure Characterization
Metric 7:

Metric 8:

Metric 9:
Metric 10:
Metric 11:
Metric 12:

Experimental System/Test Media
Preparation

Consistency of Exposure
Administration

Measurement of Test Substance
Concentration

Exposure Duration and Frequency

Number of Exposure Groups/
Spacing of Exposure Levels
Testing at or Below Solubility Limit

Low DMSO solvent was used in the preparation of the test media stock solutions, but little

other information was provided on preparation. The experimental system was 96 well
plates with one egg per plate.

High Details of exposure administration were reported and exposures were administered

consistently across study groups. 5 day exposure with one day in Hanks' buffer before
assessment.

Low Exposure concentrations were not measured or measurements were not reported.

High The duration of exposure was reported and suitable for the study type-5 day exposure

with 1 day in Hanks' buffer only.

Medium A concentration range of 2.12uM to 120uM was reported with a concentration interval
of about 0.251ogl0 units. The exact number of exposure groups was not reported.

High DMSO solvent concentration was appropriate at 0.1% (v/v)

Domain 4: Test Organism

Metric 13: Test Organism Characteristics
Metric 14: Acclimatization and Pretreatment
Conditions

High The test organisms were adequately described and were obtained from a reliable source

Low The study did not report whether test organisms were acclimatized

Continued on next page ...

Page 42 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 3479540 Table: 1 of 2

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Overall Duration: 4 - 10 days; Exposure Duration: 4 -10 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ Embryo
Development/Growth
Tris(2-chloroethyl) phosphate (TCEP)

3479540

Domain

Metric

Rating

Comments

Metric 15:

Number of Organisms and
Replicates per Group

Medium

The numbers of test organisms and replicates were reported and sufficient to character-
ize toxicological effect. 1 embryo per well/test concentration with at least 4 replicates of
each concentration.

Domain 5: Outcome Assessment

Metric 16:

Metric 17:
Metric 18:

Adequacy of Test Conditions

Outcome Assessment Methodology
Consistency of Outcome
Assessment

Low

High
High

Environmental conditions were not sufficiently reported to evaluate if adequate. Envi-
ronmental conditions of breeding adults was reported, but conditions were not reported
for embryos.

The outcome assessment methodology reported the intended outcome of interest
Details of the outcome assessment protocol were reported, and outcomes were assessed
consistently across study groups-assessment was performed 6dpf after 1 day in Hanks'
buffer without test substance

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure

Low
Medium

The study did not provide enough information to allow a comparison of environmental
conditions or other non-treatment-related factors across study groups.

There was no information in the study to suggest differences among groups in animal
attrition or health outcomes unrelated to exposure

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods
Metric 22: Reporting of Data

Metric 23: Explanation of Unexpected Outcomes

High
Medium

High

Statistical methods were adequately described

Data for exposure-related findings were presented for each treatment was reported, but
no data regarding control performance was recorded.

There were no unexpected outcomes

Additional Comments: None

Overall Quality Determination

Medium

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December 2023

Environmental Hazard Evaluation

HERO ID: 3479540 Table: 2 of 2

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Overall Duration: 4-10 days; Exposure Duration: 4-10 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ Embryo
Mortality

Tris(2-chloroethyl) phosphate (TCEP)

3479540

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

Test substance was identified by name and CAS #

Metric 2:

Test Substance Source

Low

Test substance source was reported as Sigma-Aldrich, but it did not appear to be ana-

lyzed by the performing laboratory

Metric 3:

Test Substance Purity

High

Test substance purity was 98.95%

Domain 2: Test Design

Metric 4:

Negative Controls

High

All treated groups were compared to DMSO vehicle controls.

Metric 5:

Negative Control Response

Low

The biological response of the negative control groups was not reported. The biological

response of the positive TOCP control was reported though.

Metric 6:

Randomized Allocation

Low

Researchers did not report how organisms were allocated to study groups.

Domain 3: Exposure Characterization

Metric 7:

Experimental System/Test Media

Low

DMSO solvent was used in the preparation of the test media stock solutions, but little

Preparation

other information was provided on preparation. The experimental system was 96 well

plates with one egg per plate.

Metric 8:

Consistency of Exposure

High

Details of exposure administration were reported and exposures were administered

Administration

consistently across study groups. 5 day exposure with one day in Hanks' buffer before

assessment.

Metric 9:

Measurement of Test Substance

Low

Exposure concentrations were not measured or measurements were not reported.

Concentration

Metric 10:

Exposure Duration and Frequency

High

The duration of exposure was reported and suitable for the study type-5 day exposure

with 1 day in Hanks' buffer only.

Metric 11:

Number of Exposure Groups/

Medium

A concentration range of 2.12uM to 120uM was reported with a concentration interval

Spacing of Exposure Levels

of about 0.251ogl0 units. The exact number of exposure groups was not reported.

Metric 12:

Testing at or Below Solubility Limit

High

DMSO solvent concentration was appropriate at 0.1% (v/v)

Domain 4: Test Organism

Metric 13:

Test Organism Characteristics

High

The test organisms were adequately described and were obtained from a reliable source

Metric 14:

Acclimatization and Pretreatment

Low

The study did not report whether test organisms were acclimatized

Conditions

Metric 15:

Number of Organisms and

Medium

The numbers of test organisms and replicates were reported and sufficient to character-

Replicates per Group

ize toxicological effect. 1 embryo per well/test concentration with at least 4 replicates of

each concentration.

Continued on next page ...

Page 44 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 3479540 Table: 2 of 2

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Overall Duration: 4-10 days; Exposure Duration: 4-10 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ Embryo
Mortality

Tris(2-chloroethyl) phosphate (TCEP)

3479540

Domain

Metric

Rating

Comments

Domain 5: Outcome Assessment

Metric 16:

Metric 17:
Metric 18:

Adequacy of Test Conditions

Outcome Assessment Methodology
Consistency of Outcome
Assessment

Low

High
High

Environmental conditions were not sufficiently reported to evaluate if adequate. Envi-
ronmental conditions of breeding adults was reported, but conditions were not reported
for embryos.

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure

Low The study did not provide enough information to allow a comparison of environmental

conditions or other non-treatment-related factors across study groups.

Medium There was no information in the study to suggest differences among groups in animal

attrition or health outcomes unrelated to exposure

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods

Metric 22: Reporting of Data

Metric 23: Explanation of Unexpected Outcomes

Uninformative Statistical analysis did not appear to be performed on mortality alone. Just percent mor-

tality was reported. Furthermore, death and non-hatching were grouped together with
developmental toxicity.

Medium Data for exposure-related findings were presented for each treatment was reported, but

no data regarding control performance was recorded.

Low Variability in mortality was not provided.

Additional Comments: None

Overall Quality Determination

Uninformative

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 3014520 Table: 1 of 4

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Overall Duration: 4-10 days; Exposure Duration: 4-10 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ Larvae
Development/Growth
Tris(2-chloroethyl) phosphate (TCEP)

3014520

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

The chemical substance was identified as the organophosphate flame retardant, TCEP.

Metric 2:

Test Substance Source

High

The test substance was obtained from a reputable chemical manufacturer.

Metric 3:

Test Substance Purity

High

The test substance had a 97% purity.

Domain 2: Test Design

Metric 4:

Negative Controls

High

Study authors reported using a negative control with 10% HBSS and 0.4% DMSO.

Metric 5:

Negative Control Response

High

There were no developmental abnormalities among control fish.

Metric 6:

Randomized Allocation

Medium

Embryos were collected from multiple breeding tanks and pooled, then 1 embryo/well in

96-well plates used for exposures.

Domain 3: Exposure Characterization

Metric 7:

Experimental System/Test Media

High

The experimental system and methods for preparation of test media were described in

Preparation

adequate detail.

Metric 8:

Consistency of Exposure

High

Details were sufficient to indicate consistency of exposure over 6 days post-fertilization.

Administration

Metric 9:

Measurement of Test Substance

Medium

The researchers did not measure exposure concentrations, but renewed the non-volatile

Concentration

test substance daily.

Metric 10:

Exposure Duration and Frequency

Low

The 6-day exposures, while informative, do not qualify for early-life stage testing, which

require 30-day post hatch exposures.

Metric 11:

Number of Exposure Groups/

Medium

The test organisms were exposed to 1/2 log increments of TCEP concentrations ranging

Spacing of Exposure Levels

from 0.033-100uM.

Metric 12:

Testing at or Below Solubility Limit

High

Testing was below the water solubility for TCEP.

Domain 4: Test Organism

Metric 13:

Test Organism Characteristics

Medium

The test species is an established model for toxicity testing, so although the authors did

not report the the source of the zebrafish used in the study, results for controls indicate

that this will not have a substantial impact on results.

Metric 14:

Acclimatization and Pretreatment

N/A

The authors referenced Padilla et al., 2011 for details on acclimatization and pretreat-

Conditions

ment of the test organisms.

Metric 15:

Number of Organisms and

Low

The study used 8 fish/concentration in two replicates. While not adequate for OPPT

Replicates per Group

TG 850.1400 for fish early life-stage testing, concentration-response relationships were

observed and the number was sufficient for statistical analysis.

Continued on next page ...

Page 46 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 3014520 Table: 1 of 4

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Dishaw, L. V., Hunter, D. L., Padnos, B., Padilla, S., Stapleton, H. M. (2014). Developmental Exposure to Organophosphate Flame Retardants Elicits Overt
Toxicity and Alters Behavior in Early Life Stage Zebrafish (Danio rerio). Toxicological Sciences 142(2):445-454.

Overall Duration: 4-10 days; Exposure Duration: 4-10 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ Larvae
Development/Growth
Tris(2-chloroethyl) phosphate (TCEP)

3014520

Domain

Metric

Rating

Comments

Domain 5: Outcome Assessment

Metric 16:

Adequacy of Test Conditions

High

The larvae were maintained in glass petri dishes maintained at 26C.

Metric 17:

Outcome Assessment Methodology

High

The assessment methodology assessed developmental abnormalities after 6 days of

exposure.

Metric 18:

Consistency of Outcome

High

Outcomes were assessed consistently across exposure groups.

Assessment.

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test High

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure High

The authors did not report any variations in environmental conditions or other factors
that could impact results.

The authors did not indicate differences among groups that could influence the outcome
assessment.

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods High

Metric 22: Reporting of Data High

Metric 23: Explanation of Unexpected Outcomes High

Statistical analyses were adequately described for the study.

Data for exposure-related findings were presented for each treatment and control group.
There were no unexpected outcomes for the development study.

Additional Comments: None

Overall Quality Determination

High

Page 47 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 3014520 Table: 2 of 4

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Overall Duration: 4-10 days; Exposure Duration: 4-10 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ Larvae
Mortality

Tris(2-chloroethyl) phosphate (TCEP)

3014520

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

The chemical substance was identified as the organophosphate flame retardant, TCEP.

Metric 2:

Test Substance Source

High

The test substance was obtained from a reputable chemical manufacturer.

Metric 3:

Test Substance Purity

High

The test substance had a 97% purity.

Domain 2: Test Design

Metric 4:

Negative Controls

High

Study authors reported using a negative control with 10% HBSS and 0.4% DMSO.

Metric 5:

Negative Control Response

High

There was no mortality among control fish.

Metric 6:

Randomized Allocation

Medium

Embryos were collected from multiple breeding tanks and pooled, then 1 embryo/well in

96-well plates used for exposures.

Domain 3: Exposure Characterization

Metric 7:

Experimental System/Test Media

High

The experimental system and methods for preparation of test media were described in

Preparation

adequate detail.

Metric 8:

Consistency of Exposure

High

Details were sufficient to indicate consistency of exposure over 6 days post-fertilization.

Administration

Metric 9:

Measurement of Test Substance

Medium

The researchers did not measure exposure concentrations, but renewed the non-volatile

Concentration

test substance daily.

Metric 10:

Exposure Duration and Frequency

Low

The 6-day exposures, while informative, do not qualify for early-life stage testing, which

require 30-day post hatch exposures.

Metric 11:

Number of Exposure Groups/

Medium

The test organisms were exposed to 1/2 log increments of TCEP concentrations ranging

Spacing of Exposure Levels

from 0.033-100uM.

Metric 12:

Testing at or Below Solubility Limit

High

Testing was below the water solubility for TCEP.

Domain 4: Test Organism

Metric 13:

Test Organism Characteristics

Medium

The test species is an established model for toxicity testing, so although the authors did

not report the the source of the zebrafish used in the study, results for controls indicate

that this will not have a substantial impact on results.

Metric 14:

Acclimatization and Pretreatment

Low

The authors referenced Padilla et al., 2011 for details on acclimatization and pretreat-

Conditions

ment of the test organisms.

Metric 15:

Number of Organisms and

Low

The study used 8 fish/concentration in two replicates. While not adequate for OPPT

Replicates per Group

TG 850.1400 for fish early life-stage testing, concentration-response relationships were

observed and the number was sufficient for statistical analysis.

Domain 5: Outcome Assessment

Metric 16:

Adequacy of Test Conditions

High

The larvae were maintained in glass petri dishes maintained at 26C.

Continued on next page ...

Page 48 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 3014520 Table: 2 of 4

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Dishaw, L. V., Hunter, D. L., Padnos, B., Padilla, S., Stapleton, H. M. (2014). Developmental Exposure to Organophosphate Flame Retardants Elicits Overt
Toxicity and Alters Behavior in Early Life Stage Zebrafish (Danio rerio). Toxicological Sciences 142(2):445-454.

Overall Duration: 4-10 days; Exposure Duration: 4-10 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ Larvae
Mortality

Tris(2-chloroethyl) phosphate (TCEP)

3014520

Domain

Metric

Rating

Comments

Metric 17:
Metric 18:

Outcome Assessment Methodology
Consistency of Outcome
Assessment.

High
High

The assessment methodology assessed mortality after 6 days of exposure.
Outcomes were assessed consistently across exposure groups.

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test High

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure High

The authors did not report any variations in environmental conditions or other factors
that could impact results.

The authors did not indicate differences among groups that could influence the outcome
assessment.

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods High

Metric 22: Reporting of Data High

Metric 23: Explanation of Unexpected Outcomes High

Statistical analyses were adequately described for the study.

Data for exposure-related findings were presented for each treatment and control group.
There were no unexpected outcomes for the mortality study.

Additional Comments: None

Overall Quality Determination

High

Page 49 of 170

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December 2023

Environmental Hazard Evaluation

HERO ID: 3014520 Table: 3 of 4

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Overall Duration: 4-10 days; Exposure Duration: 4-10 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ Larvae
Nutritional and Metabolic
Tris(2-chloroethyl) phosphate (TCEP)

3014520

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1: Test Substance Identity
Metric 2: Test Substance Source

Metric 3: Test Substance Purity

High
High

High

The test substance had a 97% purity.

Domain 2: Test Design

Metric 4: Negative Controls Uninformative The metabolism study compared 1 day post fertilization and 5 day post fertilization

parent chemical to metabolites in embryos and larvae. No negative control reported.

Metric 5: Negative Control Response Uninformative The study evaluated parent chemical to metabolite at 24 hours in embryos to levels in

larvae at 5 days exposure. No negative control was used.

Metric 6: Randomized Allocation Low embryos were viewed under a dissecting microscope and only healthy embryos with

intact chorions were selected for use in experiments. This is a supplemental metabolite
study in zebraflsh embryos.

Domain 3: Exposure Characterization

Metric 7:

Experimental System/Test Media

High

The experimental system and methods for preparation of test media were described in

Preparation

adequate detail.

Metric 8:

Consistency of Exposure

High

Details were sufficient to indicate consistency of exposure over 5 days.

Administration

Metric 9:

Measurement of Test Substance

Low

Larvae were exposed to 1 uM solution of TCEP in glass petri dishes for 5 days, without

Concentration

renewal or analytical monitoring.

Metric 10:

Exposure Duration and Frequency

High

The 5-day exposure was appropriate for this metabolism study.

Metric 11:

Number of Exposure Groups/

High

Exposures were to a single concentration in larvae to evaluate metabolism, which was

Spacing of Exposure Levels

later compared with 1-day exposure in embryos.

Metric 12:

Testing at or Below Solubility Limit

High

This was a metabolism study with a single concentration (which was below the water

solubility limit of TCEP).

Domain 4: Test Organism

Metric

13:

Test Organism Characteristics

Low

The test species is an established model for toxicity testing, but the authors did not re-
port the the source of the zebraflsh used in the study.

Metric

14:

Acclimatization and Pretreatment
Conditions

Low

Controls were not used for the metabolism study, and the authors referenced Padilla et
al., 2011 for details on acclimatization and pretreatment.

Metric

15:

Number of Organisms and
Replicates per Group

Medium

The larval metabolism test had 3 replicates of 3 samples (20 fish per sample).

Continued on next page ...

Page 50 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 3014520 Table: 3 of 4

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Dishaw, L. V., Hunter, D. L., Padnos, B., Padilla, S., Stapleton, H. M. (2014). Developmental Exposure to Organophosphate Flame Retardants Elicits Overt
Toxicity and Alters Behavior in Early Life Stage Zebrafish (Danio rerio). Toxicological Sciences 142(2):445-454.

Overall Duration: 4-10 days; Exposure Duration: 4-10 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ Larvae
Nutritional and Metabolic
Tris(2-chloroethyl) phosphate (TCEP)

3014520

Domain

Metric

Rating

Comments

Domain 5: Outcome Assessment

Metric 16:

Adequacy of Test Conditions

High

The larvae were maintained in glass petri dishes maintained at 26C.

Metric 17:

Outcome Assessment Methodology

High

The assessment methodology assessed metabolism after 5 days of exposure.

Metric 18:

Consistency of Outcome

High

Outcomes were assessed consistently across exposure groups.

Assessment

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure

High
High

The authors did not report any variations in environmental conditions or other factors
that could impact results.

The authors did not indicate differences among groups that could influence the outcome
assessment.

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods High Tissue concentrations of the parent and primary metabolites were averaged, along with

a two-way ANOVA and Sidak multiple comparisons for both the embryo and larval
metabolism tests.

Metric 22: Reporting of Data High Data for the metabolism study for embryos and larva were presented.

Metric 23: Explanation of Unexpected Outcomes High There were no unexpected outcomes for the metabolism study.

Additional Comments: Metabolism study for zebrafish embryos accompanied by the larval metabolism study.

Overall Quality Determination Uninformative

Page 51 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 3014520 Table: 4 of 4

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Overall Duration: 4-10 days; Exposure Duration: 4-10 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ Larvae
Behavioral

Tris(2-chloroethyl) phosphate (TCEP)

3014520

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

The chemical substance was identified as the organophosphate flame retardant, TCEP.

Metric 2:

Test Substance Source

High

The test substance was obtained from a reputable chemical manufacturer.

Metric 3:

Test Substance Purity

High

The test substance had a 97% purity.

Domain 2: Test Design

Metric 4:

Negative Controls

High

Study authors reported using a negative control with 10% HBSS and 0.4% DMSO.

Metric 5:

Negative Control Response

High

The control larval swimming behavior appears acceptable.

Metric 6:

Randomized Allocation

Medium

Embryos were extracted from multiple breeding tanks and pooled before cleaning and

inspection for viability.

Domain 3: Exposure Characterization

Metric 7:

Experimental System/Test Media

High

The experimental system and methods for preparation of test media were described in

Preparation

adequate detail.

Metric 8:

Consistency of Exposure

High

Details were sufficient to indicate consistency of exposure over 6 days post-fertilization.

Administration

Metric 9:

Measurement of Test Substance

Medium

The researchers did not measure exposure concentrations, but renewed the non-volatile

Concentration

test substance daily.

Metric 10:

Exposure Duration and Frequency

Low

The 6-day exposures, while informative, do not qualify for early-life stage testing, which

require 30-day post hatch exposures.

Metric 11:

Number of Exposure Groups/

Medium

The test organisms were exposed to 5 concentrations of 1/4 log increments of TCEP

Spacing of Exposure Levels

concentrations set below concentrations that resulted in mortality or developmental

abnormalities.

Metric 12:

Testing at or Below Solubility Limit

High

Testing was below the water solubility for TCEP.

Domain 4: Test Organism

Metric 13:

Test Organism Characteristics

Medium

The test species is an established model for toxicity testing, so although the authors did

not report the the source of the zebrafish used in the study, results for controls indicate

that this will not have a substantial impact on results.

Metric 14:

Acclimatization and Pretreatment

Low

The authors referenced Padilla et al., 2011 for details on acclimatization and pretreat-

Conditions

ment of the test organisms.

Metric 15:

Number of Organisms and

Low

The study used 6-12 fish/concentration with 2-4 replicates. While not adequate for

Replicates per Group

OPPT TG 850.1400 for fish early life-stage testing, concentration-response relationships

were observed and the number was sufficient for statistical analysis.

Domain 5: Outcome Assessment

Continued on next page ...

Page 52 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 3014520 Table: 4 of 4

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Dishaw, L. V., Hunter, D. L., Padnos, B., Padilla, S., Stapleton, H. M. (2014). Developmental Exposure to Organophosphate Flame Retardants Elicits Overt
Toxicity and Alters Behavior in Early Life Stage Zebrafish (Danio rerio). Toxicological Sciences 142(2):445-454.

Overall Duration: 4-10 days; Exposure Duration: 4-10 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ Larvae
Behavioral

Tris(2-chloroethyl) phosphate (TCEP)

3014520

Domain

Metric

Rating

Comments

Metric 16:
Metric 17:

Metric 18:

Adequacy of Test Conditions
Outcome Assessment Methodology

Consistency of Outcome
Assessment

High
High

High

The larvae were maintained in glass petri dishes maintained at 26C.

The assessment methodology assessed changes in larval swimming behavior after 6 days

of exposure.

Outcomes were assessed consistently across exposure groups.

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure

High
High

The authors did not report any variations in environmental conditions or other factors
that could impact results.

The authors did not indicate differences among groups that could influence the outcome

assessment.

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods High

Metric 22: Reporting of Data High

Metric 23: Explanation of Unexpected Outcomes High

Statistical analyses were adequately described for the study.

Data for exposure-related findings were presented for each treatment and control group.
There were no unexpected outcomes for the behavioral study.

Additional Comments: None

Overall Quality Determination

High

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December 2023

Tris(2-chloroethyl) phosphate (TCEP) Environmental Hazard Evaluation HERO ID: 4180931 Table: 1 of 1

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Overall Duration: 4-10 days; Exposure Duration: 4 - 10 days

Aquatic (freshwater); Cell Culture Media; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact
uptake route)

Vertebrate; Fish; Danio rerio\ wild type; Larvae
Behavioral

Tris(2-chloroethyl) phosphate (TCEP)

4180931

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1: Test Substance Identity
Metric 2: Test Substance Source

Metric 3: Test Substance Purity

High Well documented in supplemental data and table 1. IUPAC name and structure provided.
Low NTP is listed as supplier, Sigma-Aldrich as the manufacturer, purity was not analytically
verified.

High Information is in supplemental data, listed as 99% pure.

Domain 2: Test Design

Metric 4: Negative Controls
Metric 5: Negative Control Response
Metric 6: Randomized Allocation

High DMSO vehicle control was used as negative control, and chlorpyrifos or heptachlor were
used as positive controls.

Low No report on survival or malformation rate for controls, no comparison between clean
and solvent controls were reported.

Domain 3: Exposure Characterization

Metric 7:

Experimental System/Test Media

High

Experimental system was explained in detail.

Preparation

Metric 8:

Consistency of Exposure

High

Method for administering the test substance was reported in detail, and was consistent

Administration

across study groups.

Metric 9:

Measurement of Test Substance

Low

Test substance concentrations were not analytically verified. Wells were covered and

Concentration

sealed with Parafilm to minimize evaporation.

Metric 10:

Exposure Duration and Frequency

High

durations were based on other studies

Metric 11:

Number of Exposure Groups/

High

Five concentrations were tested, covering an adequate range for a response. A range

Spacing of Exposure Levels

finding study was conducted to set the highest dose that would not cause any acute ef-

fects or lethality that would confound the behavioral assessment.

Metric 12:

Testing at or Below Solubility Limit

Medium

No indication given for how solvent concentrations were chosen.

Domain 4: Test Organism

Metric 13: Test Organism Characteristics

Continued on next page ...

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 4180931 Table: 1 of 1

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Overall Duration: 4-10 days; Exposure Duration: 4 - 10 days

Aquatic (freshwater); Cell Culture Media; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact
uptake route)

Vertebrate; Fish; Danio rerio\ wild type; Larvae
Behavioral

Tris(2-chloroethyl) phosphate (TCEP)

4180931

Domain

Metric

Rating

Comments

Metric 14:
Metric 15:

Acclimatization and Pretreatment
Conditions

Number of Organisms and
Replicates per Group

High
Medium

Treatments were reported as similar across all treatments.

The number tested at each concentration were reported - 24 larvae per dose.

Domain 5: Outcome Assessment

Metric 16: Adequacy of Test Conditions

Metric 17: Outcome Assessment Methodology
Metric 18: Consistency of Outcome
Assessment.

Medium Conditions seemed adequate for embryo development, small quantities are difficult to

assess.

High Well documented assessment procedure.

High No deviations in assessment procedures were reported.

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure

High No confounding variables were reported.

Medium No unrelated outcomes reported downgraded because only normal larvae were assessed.

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods

Metric 22: Reporting of Data

Metric 23: Explanation of Unexpected Outcomes

High Well documented analysis methods
High All behavioral data was presented in a reasonable manner.

Medium Downgraded because they only assessed normal larvae and none during the acclimation
phase.

Additional Comments: None

Overall Quality Determination

High

Page 55 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469290 Table: 1 of 5

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Li, R., Wang, H., Mi, C., Feng, C., Zhang, L., Yang, L., Zhou, B. (2019). The adverse effect of TCIPP and TCEP on neurodevelopment of zebrafish

embryos/larvae. Chemosphere 220811-817.

Overall Duration: 4-10 days; Exposure Duration: 4-10 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ AB strain; Embryo
Reproductive/Teratogenic
Tris(2-chloroethyl) phosphate (TCEP)

5469290

Domain

Metric

Rating

Comments

Domain 1: Test Substance







Metric 1:

Test Substance Identity

High

TCEP identified by CAS number and name

Metric 2:

Test Substance Source

Low

TCEP was not analytically verified

Metric 3:

Test Substance Purity

Medium

97% purity reported

Domain 2: Test Design







Metric 4:

Negative Controls

Low

the solvent concentration of treatments wasn't reported

Metric 5:

Negative Control Response

High

good survival (>90% ) in controls

Metric 6:

Randomized Allocation

Low

did not report random allocation

Domain 3: Exposure Characterization







Metric 7:

Experimental System/Test Media

High

adequate test system and description



Preparation





Metric 8:

Consistency of Exposure

High

no inconsistencies reported between treatments



Administration





Metric 9:

Measurement of Test Substance

High

test concentrations were measured



Concentration





Metric 10:

Exposure Duration and Frequency

High

adequate duration to observe desired effects

Metric 11:

Number of Exposure Groups/

High

adequate number of exposure groups to assess outcome



Spacing of Exposure Levels





Metric 12:

Testing at or Below Solubility Limit

High

well within solubility of TCEP

Domain 4: Test Organism







Metric 13:

Test Organism Characteristics

High

organisms were adequately characterized

Metric 14:

Acclimatization and Pretreatment

High

consistent pre treatment across all concentrations



Conditions





Metric 15:

Number of Organisms and

Medium

four replicates were used



Replicates per Group





Domain 5: Outcome Assessment







Metric 16:

Adequacy of Test Conditions

Medium

test water parameters except temperature were not reported

Metric 17:

Outcome Assessment Methodology

Medium

adequate to determine percent hatch and survival

Metric 18:

Consistency of Outcome

High

no inconsistencies were reported

Assessment.

Continued on next page ...

Page 56 of 170

-------
Tris(2-chloroethyl) phosphate (TCEP)

PUBLIC RELEASE DRAFT - DO NOT CITE OR QUOTE
December 2023

Environmental Hazard Evaluation

HERO ID: 5469290 Table: 1 of 5

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

... continued from previous page

Li, R., Wang, H., Mi, C., Feng, C., Zhang, L., Yang, L., Zhou, B. (2019). The adverse effect of TCIPP and TCEP on neurodevelopment of zebrafish

embryos/larvae. Chemosphere 220811-817.

Overall Duration: 4-10 days; Exposure Duration: 4-10 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ AB strain; Embryo
Reproductive/Teratogenic
Tris(2-chloroethyl) phosphate (TCEP)

5469290

Domain

Metric

Rating

Comments

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure

High no confounding variables were reported

Medium There was no information in the study to suggest differences among groups in animal
attrition or health outcomes unrelated to exposure (e.g., infection) that could influence
the outcome assessment.

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods	High	statistical methodology was adequate for this outcome

Metric 22: Reporting of Data	High	data reporting was reasonably clear

Metric 23: Explanation of Unexpected Outcomes	High	no unexpected outcomes were reported

Additional Comments: This evaluation form pertains to hatching rate of embryos following exposure to 0, 100, 500 or 2500 mg/L of TCEP.

Overall Quality Determination	High

Page 57 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469290 Table: 2 of 5

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Li, R., Wang, H., Mi, C., Feng, C., Zhang, L., Yang, L., Zhou, B. (2019). The adverse effect of TCIPP and TCEP on neurodevelopment of zebrafish

embryos/larvae. Chemosphere 220811-817.

Overall Duration: 4-10 days; Exposure Duration: 4-10 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)
Vertebrate; Fish; Danio rerio\ AB strain; Embryo

Mechanistic-Biomarkers (exposure and effect)-Cell signaling/function-Neurotoxicology

Tris(2-chloroethyl) phosphate (TCEP)

5469290

Domain

Metric

Rating

Comments

Domain 1: Test Substance







Metric 1:

Test Substance Identity

High

TCEP identified by CAS number and name

Metric 2:

Test Substance Source

Low

TCEP was not analytically verified

Metric 3:

Test Substance Purity

Medium

97% purity reported

Domain 2: Test Design







Metric 4:

Negative Controls

Low

the solvent concentration of treatments wasn't reported

Metric 5:

Negative Control Response

High

good survival (>90% ) in controls

Metric 6:

Randomized Allocation

Low

did not report random allocation

Domain 3: Exposure Characterization







Metric 7:

Experimental System/Test Media

High

adequate test system and description



Preparation





Metric 8:

Consistency of Exposure

High

no inconsistencies reported between treatments



Administration





Metric 9:

Measurement of Test Substance

High

test concentrations were measured



Concentration





Metric 10:

Exposure Duration and Frequency

Medium

adequate duration to observe desired effects

Metric 11:

Number of Exposure Groups/

High

the number of exposure groups were adequate to assess this outcome



Spacing of Exposure Levels





Metric 12:

Testing at or Below Solubility Limit

High

well within solubility of TCEP

Domain 4: Test Organism







Metric 13:

Test Organism Characteristics

High

organisms were adequately characterized

Metric 14:

Acclimatization and Pretreatment

High

consistent pre treatment across all concentrations



Conditions





Metric 15:

Number of Organisms and

Medium

four replicates were used



Replicates per Group





Domain 5: Outcome Assessment







Metric 16:

Adequacy of Test Conditions

Medium

test water parameters except temperature were not reported

Metric 17:

Outcome Assessment Methodology

Medium

enzyme and protein determination and gene transcription methods seemed sound

Metric 18:

Consistency of Outcome

High

no inconsistencies were reported



Assessment





Domain 6: Confounding / Variable Control

Continued on next page .

Page 58 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469290 Table: 2 of 5

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

... continued from previous page

Li, R., Wang, H., Mi, C., Feng, C., Zhang, L., Yang, L., Zhou, B. (2019). The adverse effect of TCIPP and TCEP on neurodevelopment of zebrafish

embryos/larvae. Chemosphere 220811-817.

Overall Duration: 4-10 days; Exposure Duration: 4-10 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)
Vertebrate; Fish; Danio rerio\ AB strain; Embryo

Mechanistic-Biomarkers (exposure and effect)-Cell signaling/function-Neurotoxicology

Tris(2-chloroethyl) phosphate (TCEP)

5469290

Domain

Metric

Rating

Comments

Metric 19: Confounding Variables in Test

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure

High no confounding variables were reported

Medium There was no information in the study to suggest differences among groups in animal
attrition or health outcomes unrelated to exposure (e.g., infection) that could influence
the outcome assessment.

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods	High	statistical methodology was adequate for this outcome

Metric 22: Reporting of Data	High	determinations of levels of significance seemed sound

Metric 23: Explanation of Unexpected Outcomes	High	no unexpected outcomes were reported

Additional Comments: This evaluation form pertains to mechanistic endpoints (neurotransmitter contents, AchE activity, gene transcription and protein expression) evaluated in
	zebrafish larvae after exposure to TCEP.	

Overall Quality Determination	High

Page 59 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469290 Table: 3 of 5

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Li, R., Wang, H., Mi, C., Feng, C., Zhang, L., Yang, L., Zhou, B. (2019). The adverse effect of TCIPP and TCEP on neurodevelopment of zebrafish

embryos/larvae. Chemosphere 220811-817.

Overall Duration: 4-10 days; Exposure Duration: 4-10 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ AB strain; Embryo
Behavioral

Tris(2-chloroethyl) phosphate (TCEP)

5469290

Domain

Metric

Rating

Comments

Domain 1: Test Substance







Metric 1:

Test Substance Identity

High

TCEP identified by CAS number and name

Metric 2:

Test Substance Source

Low

TCEP was not analytically verified

Metric 3:

Test Substance Purity

Medium

97% purity reported for TCEP

Domain 2: Test Design







Metric 4:

Negative Controls

Low

the solvent concentration of treatments wasn't reported

Metric 5:

Negative Control Response

High

good survival (>90% ) in controls

Metric 6:

Randomized Allocation

Low

did not report random allocation

Domain 3: Exposure Characterization







Metric 7:

Experimental System/Test Media

High

adequate test system and description



Preparation





Metric 8:

Consistency of Exposure

High

no inconsistencies reported between treatments



Administration





Metric 9:

Measurement of Test Substance

High

test concentrations were measured



Concentration





Metric 10:

Exposure Duration and Frequency

High

adequate duration to observe desired effects

Metric 11:

Number of Exposure Groups/

High

adequate number of organisms to observe desired effects



Spacing of Exposure Levels





Metric 12:

Testing at or Below Solubility Limit

High

well within solubility of TCEP

Domain 4: Test Organism







Metric 13:

Test Organism Characteristics

High

adequately documented organism characteristics

Metric 14:

Acclimatization and Pretreatment

High

consistent pre treatment across all concentrations



Conditions





Metric 15:

Number of Organisms and

Medium

four replicates were used



Replicates per Group





Domain 5: Outcome Assessment







Metric 16:

Adequacy of Test Conditions

Medium

test water parameters except temperature were not reported

Metric 17:

Outcome Assessment Methodology

Medium

adequate measurements but excluding malformed organisms is concerning

Metric 18:

Consistency of Outcome

High

no inconsistencies were reported



Assessment





Domain 6: Confounding / Variable Control

Continued on next page .

Page 60 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469290 Table: 3 of 5

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Li, R., Wang, H., Mi, C., Feng, C., Zhang, L., Yang, L., Zhou, B. (2019). The adverse effect of TCIPP and TCEP on neurodevelopment of zebrafish

embryos/larvae. Chemosphere 220811-817.

Overall Duration: 4-10 days; Exposure Duration: 4-10 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ AB strain; Embryo
Behavioral

Tris(2-chloroethyl) phosphate (TCEP)

5469290

Domain

Metric

Rating

Comments

Metric 19:
Metric 20:

Confounding Variables in Test
Design and Procedures
Outcomes Unrelated to Exposure

High
Medium

no confounding variables were reported

There was no information in the study to suggest differences among groups in animal
attrition or health outcomes unrelated to exposure (e.g., infection) that could influence
the outcome assessment.

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods

Metric 22: Reporting of Data

Metric 23: Explanation of Unexpected Outcomes

High
High
High

statistical methodology seemed sound
data reporting was reasonably clear

adequate explanation of difference between light and dark response

Additional Comments: This evaluation form pertains to locomotor behavior measurement of zebrafish larvae.

Overall Quality Determination

High

Page 61 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469290 Table: 4 of 5

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Li, R., Wang, H., Mi, C., Feng, C., Zhang, L., Yang, L., Zhou, B. (2019). The adverse effect of TCIPP and TCEP on neurodevelopment of zebrafish

embryos/larvae. Chemosphere 220811-817.

Overall Duration: 4-10 days; Exposure Duration: 4-10 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ AB strain; Embryo
Development/Growth
Tris(2-chloroethyl) phosphate (TCEP)

5469290

Domain

Metric

Rating

Comments

Domain 1: Test Substance







Metric 1:

Test Substance Identity

High

TCEP identified by CAS number and name

Metric 2:

Test Substance Source

Low

TCEP was not analytically verified

Metric 3:

Test Substance Purity

Medium

97% purity reported for TCEP

Domain 2: Test Design







Metric 4:

Negative Controls

Low

the solvent concentration of treatments wasn't reported

Metric 5:

Negative Control Response

High

good survival (>90% ) in controls

Metric 6:

Randomized Allocation

Low

did not report random allocation

Domain 3: Exposure Characterization







Metric 7:

Experimental System/Test Media

High

adequate test system and description



Preparation





Metric 8:

Consistency of Exposure

High

no inconsistencies reported between treatments



Administration





Metric 9:

Measurement of Test Substance

High

test concentrations were reported



Concentration





Metric 10:

Exposure Duration and Frequency

Medium

adequate duration to observe desired effects, not primary purpose of study

Metric 11:

Number of Exposure Groups/

High

adequate number of exposure groups to assess outcomes



Spacing of Exposure Levels





Metric 12:

Testing at or Below Solubility Limit

High

well within solubility of TCEP

Domain 4: Test Organism







Metric 13:

Test Organism Characteristics

High

adequate documentation of organism characteristics

Metric 14:

Acclimatization and Pretreatment

High

consistent pre treatment across all concentrations



Conditions





Metric 15:

Number of Organisms and

Medium

four replicates were used



Replicates per Group





Domain 5: Outcome Assessment







Metric 16:

Adequacy of Test Conditions

Medium

test water parameters except temperature were not reported

Metric 17:

Outcome Assessment Methodology

Medium

type of malformations not reported

Metric 18:

Consistency of Outcome

High

no inconsistencies were noted



Assessment





Domain 6: Confounding / Variable Control

Continued on next page .

Page 62 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469290 Table: 4 of 5

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

... continued from previous page

Li, R., Wang, H., Mi, C., Feng, C., Zhang, L., Yang, L., Zhou, B. (2019). The adverse effect of TCIPP and TCEP on neurodevelopment of zebrafish

embryos/larvae. Chemosphere 220811-817.

Overall Duration: 4-10 days; Exposure Duration: 4-10 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ AB strain; Embryo
Development/Growth
Tris(2-chloroethyl) phosphate (TCEP)

5469290

Domain

Metric

Rating

Comments

Metric 19: Confounding Variables in Test

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure

High no unexpected variables were reported

Medium There was no information in the study to suggest differences among groups in animal
attrition or health outcomes unrelated to exposure (e.g., infection) that could influence
the outcome assessment.

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods	High	statistical methods were adequately explained

Metric 22: Reporting of Data	High	documentation of data reporting as adequate

Metric 23: Explanation of Unexpected Outcomes	High	no unexplained outcomes were reported

Additional Comments: This evaluation form pertains to malformation rate of embryos following exposure to 0, 100, 500 or 2500 mg/L of TCEP.

Overall Quality Determination	High

Page 63 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469290 Table: 5 of 5

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Li, R., Wang, H., Mi, C., Feng, C., Zhang, L., Yang, L., Zhou, B. (2019). The adverse effect of TCIPP and TCEP on neurodevelopment of zebrafish

embryos/larvae. Chemosphere 220811-817.

Overall Duration: 4-10 days; Exposure Duration: 4-10 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ AB strain; Embryo
Mortality

Tris(2-chloroethyl) phosphate (TCEP)

5469290

Domain

Metric

Rating

Comments

Domain 1: Test Substance







Metric 1:

Test Substance Identity

High

TCEP identified by CAS number and name

Metric 2:

Test Substance Source

Low

TCEP was not analytically verified

Metric 3:

Test Substance Purity

Medium

97% purity reported

Domain 2: Test Design







Metric 4:

Negative Controls

Low

the solvent concentration of treatments wasn't reported

Metric 5:

Negative Control Response

High

good survival (>90% ) in controls

Metric 6:

Randomized Allocation

Low

did not report random allocation

Domain 3: Exposure Characterization







Metric 7:

Experimental System/Test Media

High

adequate test system and description



Preparation





Metric 8:

Consistency of Exposure

High

no inconsistencies reported between treatments



Administration





Metric 9:

Measurement of Test Substance

High

test concentrations were measured



Concentration





Metric 10:

Exposure Duration and Frequency

High

adequate duration to observe desired effects

Metric 11:

Number of Exposure Groups/

High

adequate number of exposure groups to assess outcome



Spacing of Exposure Levels





Metric 12:

Testing at or Below Solubility Limit

High

well within solubility of TCEP

Domain 4: Test Organism







Metric 13:

Test Organism Characteristics

High

organisms were adequately characterized

Metric 14:

Acclimatization and Pretreatment

High

consistent pre treatment across all concentrations



Conditions





Metric 15:

Number of Organisms and

Medium

four replicates were used



Replicates per Group





Domain 5: Outcome Assessment







Metric 16:

Adequacy of Test Conditions

Medium

test water parameters except temperature were not reported

Metric 17:

Outcome Assessment Methodology

Medium

adequate to determine percent hatch and survival

Metric 18:

Consistency of Outcome

High

no inconsistencies were reported



Assessment





Domain 6: Confounding / Variable Control

Continued on next page .

Page 64 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469290 Table: 5 of 5

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Li, R., Wang, H., Mi, C., Feng, C., Zhang, L., Yang, L., Zhou, B. (2019). The adverse effect of TCIPP and TCEP on neurodevelopment of zebrafish

embryos/larvae. Chemosphere 220811-817.

Overall Duration: 4-10 days; Exposure Duration: 4-10 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ AB strain; Embryo
Mortality

Tris(2-chloroethyl) phosphate (TCEP)

5469290

Domain



Metric

Rating Comments



Metric 19:

Confounding Variables in Test

High no confounding variables were reported





Design and Procedures





Metric 20:

Outcomes Unrelated to Exposure

High no unrelated outcomes reported

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods

Metric 22: Reporting of Data

Metric 23: Explanation of Unexpected Outcomes

High statistical methodology was adequate for this outcome
High data reporting was reasonably clear
High no unexpected outcomes were reported

Additional Comments: None

Overall Quality Determination

High

Page 65 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 2953504 Table: 1 of 3

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Overall Duration: 4-10 days; Exposure Duration: 4-10 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ wild type (Tropical 5D); Embryo
Behavioral

Tris(2-chloroethyl) phosphate (TCEP)

2953504

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

The chemical is referred to by name, structure, and CASRN.

Metric 2:

Test Substance Source

Low

Source was reported but the authors did not perform analytical verification.

Metric 3:

Test Substance Purity

High

Purity is >98% and reported in Table 3.

Domain 2: Test Design

Metric 4: Negative Controls
Metric 5: Negative Control Response
Metric 6: Randomized Allocation

High A solvent control (DMSO at 0.64% ) was used.

High All control responses are reported in the supplemental data PDF.

Domain 3: Exposure Characterization

Metric 7:

Experimental System/Test Media

Medium

Preparation

Metric 8:

Consistency of Exposure

High

Administration

Metric 9:

Measurement of Test Substance

Low

Concentration

Metric 10:

Exposure Duration and Frequency

High

Metric 11:

Number of Exposure Groups/

High

Spacing of Exposure Levels

Metric 12:

Testing at or Below Solubility Limit

Medium

The exposures were static non-renewal from 6 to 120 hpf. Renewal of compounds
would have been preferred.

Exposures were conducted consistently among treatments and control.

No verification was performed, concentrations are reported as nominal.

Exposure was initiated at 6 hpf and continued till 120 hpf. The assessments at 24 and
120 hpf are appropriate to capture embryo and larval periods.

Concentrations were: 64, 6.4, 0.64, 0.064, 0.0064, and 0 uM and represent a broad range
of exposure concentrations.

Domain 4: Test Organism

Metric 13: Test Organism Characteristics

High The strain and source for broodfish was reported.

Continued on next page ...

Page 66 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 2953504 Table: 1 of 3

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Overall Duration: 4-10 days; Exposure Duration: 4-10 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ wild type (Tropical 5D); Embryo
Behavioral

Tris(2-chloroethyl) phosphate (TCEP)

2953504

Domain

Metric

Rating

Comments

Metric 14:
Metric 15:

Acclimatization and Pretreatment
Conditions

Number of Organisms and
Replicates per Group

High
Medium

Water quality parameters were well described and accordance with protocols under
Oregon State University"s Institutional Animal Care and Use Committee.

All sample size numbers for treatment and control groups are listed in the first two pages
of the supplemental data PDF for this work.

Domain 5: Outcome Assessment

Metric 16:

Adequacy of Test Conditions

Medium

Photoperiod and temperature were reported, while other water quality conditions were
not.

Movement assays for embryos (24 hpf) and larvae (120 hpf) were described in detail on
page 7 of 19.

Assessment was consistent among treatments and control.

Metric 17:
Metric 18:

Outcome Assessment Methodology

Consistency of Outcome
Assessment.

Medium
High

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure

High Nothing was reported to indicate differences in study groups based on environmental
factors.

Medium No information in the study to indicate that differences were from animal attrition or
health outcomes.

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods

Metric 22: Reporting of Data

Metric 23: Explanation of Unexpected Outcomes

High All raw data across time for embryo and larval movement is reported in the supplemen-
tal notes (24 and 120 hpf).

High No unexpected outcomes were reported by the authors.

Additional Comments: This form is for embryo movement assessment at 120 hpf for TCEP.

Continued on next page ...

Page 67 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 2953504 Table: 1 of 3

... continued from previous page

Study Citation:

Noyes, P. D., Haggard, D. E., Gonnerman, G. D., Tanguay, R. L. (2015). Advanced morphological - behavioral test platform reveals neurodevelopmental
defects in embryonic zebrafish exposed to comprehensive suite of halogenated and organophosphate flame retardants. Toxicological Sciences 145(1):177-
195.

Overall Duration: 4-10 days; Exposure Duration: 4-10 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ wild type (Tropical 5D); Embryo
Behavioral

Tris(2-chloroethyl) phosphate (TCEP)

2953504

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Domain

Metric Rating Comments

Overall Quality Determination High



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-------
Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 2953504 Table: 2 of 3

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Overall Duration: 4-10 days; Exposure Duration: 4-10 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ wild type (Tropical 5D); Embryo
Mortality

Tris(2-chloroethyl) phosphate (TCEP)

2953504

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

The chemical is referred to by name, structure, and CASRN.

Metric 2:

Test Substance Source

Low

Source was reported but the authors did not perform analytical verification.

Metric 3:

Test Substance Purity

High

Purity is >98% and reported in Table 3.

Domain 2: Test Design

Metric 4: Negative Controls
Metric 5: Negative Control Response
Metric 6: Randomized Allocation

High A solvent control (DMSO at 0.64% ) was used.

High All control responses are reported in the supplemental data PDF.

Domain 3: Exposure Characterization

Metric 7:

Experimental System/Test Media

Medium

Preparation

Metric 8:

Consistency of Exposure

High

Administration

Metric 9:

Measurement of Test Substance

Low

Concentration

Metric 10:

Exposure Duration and Frequency

High

Metric 11:

Number of Exposure Groups/

High

Spacing of Exposure Levels

Metric 12:

Testing at or Below Solubility Limit

Medium

would have been preferred.

Exposures were conducted consistently among treatments and control.

No verification was performed, concentrations are reported as nominal.

Exposure was initiated at 6 hpf and continued till 120 hpf. The assessments at 24 and
120 hpf are appropriate to capture embryo and larval periods.

Concentrations were: 64, 6.4, 0.64, 0.064, 0.0064, and 0 uM and represent a broad range
of exposure concentrations.

Domain 4: Test Organism

Metric 13: Test Organism Characteristics High

Metric 14: Acclimatization and Pretreatment High

Conditions

The strain and source for broodfish was reported.

Water quality parameters were well described and accordance with protocols under
Oregon State University"s Institutional Animal Care and Use Committee.

Continued on next page ...

Page 69 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 2953504 Table: 2 of 3

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Overall Duration: 4-10 days; Exposure Duration: 4-10 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ wild type (Tropical 5D); Embryo
Mortality

Tris(2-chloroethyl) phosphate (TCEP)

2953504

Domain

Metric

Rating

Comments

Metric 15:

Number of Organisms and
Replicates per Group

Medium

All sample size numbers for treatment and control groups are listed in the first two pages
of the supplemental data PDF for this work.

Domain 5: Outcome Assessment

Metric 16:

Adequacy of Test Conditions

Medium

Photoperiod and temperature were reported, while other water quality conditions were
not.

Mortality assessment was described in the section titled "Developmental malformation
evaluations" but authors did not report specific criteria for death (ie, movement, heart-
beat, color, etc.).

Assessment was consistent among treatments and control.

Metric 17:
Metric 18:

Outcome Assessment Methodology

Consistency of Outcome
Assessment.

Medium
High

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure

High Nothing was reported to indicate differences in study groups based on environmental
factors.

Medium No information in the study to indicate that differences were from animal attrition or
health outcomes.

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods

Metric 22: Reporting of Data

Metric 23: Explanation of Unexpected Outcomes

High All counts of data per treatment and control group are represented in the supplemental
data for each compound and time point (24 and 120 hpf).

High No unexpected outcomes were reported by the authors.

Additional Comments:

This form is for Mortality assessment at 120 hpf for TCEP. The specific data and statistical significance for each compound are located within the
supplemental PDF.

Continued on next page ...

Page 70 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 2953504 Table: 2 of 3

... continued from previous page

Study Citation:

Noyes, P. D., Haggard, D. E., Gonnerman, G. D., Tanguay, R. L. (2015). Advanced morphological - behavioral test platform reveals neurodevelopmental
defects in embryonic zebrafish exposed to comprehensive suite of halogenated and organophosphate flame retardants. Toxicological Sciences 145(1):177-
195.

Overall Duration: 4-10 days; Exposure Duration: 4-10 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ wild type (Tropical 5D); Embryo
Mortality

Tris(2-chloroethyl) phosphate (TCEP)

2953504

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Domain

Metric Rating Comments

Overall Quality Determination High



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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 2953504 Table: 3 of 3

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Noyes, P. D., Haggard, D. E., Gonnerman, G. D., Tanguay, R. L. (2015). Advanced morphological - behavioral test platform reveals neurodevelopmental
defects in embryonic zebrafish exposed to comprehensive suite of halogenated and organophosphate flame retardants. Toxicological Sciences 145(1):177-
195.

Overall Duration: 4-10 days; Exposure Duration: 4-10 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ wild type (Tropical 5D); Embryo
Development/Growth
Tris(2-chloroethyl) phosphate (TCEP)

2953504

Domain

Metric

Rating

Comments

Domain 1: Test Substance







Metric 1:

Test Substance Identity

High

The chemical is referred to by name, structure, and CASRN.

Metric 2:

Test Substance Source

Low

Source was reported but the authors did not perform analytical verification.

Metric 3:

Test Substance Purity

High

Purity is >98% and reported in Table 3.

Domain 2: Test Design

Metric 4: Negative Controls
Metric 5: Negative Control Response
Metric 6: Randomized Allocation

High A solvent control (DMSO at 0.64% ) was used.

High All control responses are reported in the supplemental data PDF.

Medium Although not specifically reported as "random" the process of chorion removal and
distribution into well plates was automated and is detailed in: "Mandrell, David, Lisa
Truong, Caleb Jephson, Mushfiqur R. Sarker, Aaron Moore, Christopher Lang, Michael
T. Simonich, and Robert L. Tanguay. "Automated zebrafish chorion removal and single
embryo placement: optimizing throughput of zebrafish developmental toxicity screens."
Journal of laboratory automation 17, no. 1 (2012): 66-74."

Domain 3: Exposure Characterization

Metric 7:

Experimental System/Test Media

Medium



Preparation



Metric 8:

Consistency of Exposure

High



Administration



Metric 9:

Measurement of Test Substance

Low



Concentration



Metric 10:

Exposure Duration and Frequency

High

Metric 11: Number of Exposure Groups/
Spacing of Exposure Levels

High

would have been preferred.

Exposures were conducted consistently among treatments and control.

No verification was performed, concentrations are reported as nominal.

Exposure was initiated at 6 hpf and continued till 120 hpf. The assessments at 24 and
120 hpf are appropriate to capture embryo and larval periods. 24 hour developmental
assessments included: delays in developmental progression, notochord deformities, and
altered spontaneous movements. 120 hour developmental assessments included: 17 de-
velopmental malformations, including yolk sac edema (YSE) and pericardial edema
(PE); body axis (AXIS), trunk length (TRUN), caudal fin (CFIN), pectoral fin (PFIN),
pigmentation (PIG), and somite (SOMI) deformities; eye (EYE), snout (SNOU), jaw
(JAW), and otolith (OTIC) malformations; gross brain development (BRAIN); noto-
chord (NC) and circulatory (CIRC) deformities; swim bladder presence and inflation
(SWIM); and touch responses (TR).

Concentrations were: 64, 6.4, 0.64, 0.064, 0.0064, and 0 uM and represent a broad range
of exposure concentrations.

Continued on next page ...

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Tris(2-chloroethyl) phosphate (TCEP)

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Environmental Hazard Evaluation

HERO ID: 2953504 Table: 3 of 3

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Overall Duration: 4-10 days; Exposure Duration: 4-10 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ wild type (Tropical 5D); Embryo
Development/Growth
Tris(2-chloroethyl) phosphate (TCEP)

2953504

Domain

Metric

Rating

Comments

Metric 12:

Testing at or Below Solubility Limit

Medium

Domain 4: Test Organism

Metric 13:
Metric 14:

Metric 15:

Test Organism Characteristics
Acclimatization and Pretreatment
Conditions

Number of Organisms and
Replicates per Group

High
High

Medium

The strain and source for broodfish was reported.

Water quality parameters were well described and accordance with protocols under
Oregon State University"s Institutional Animal Care and Use Committee.

All sample size numbers for treatment and control groups are listed in the first two pages
of the supplemental data PDF for this work.

Domain 5: Outcome Assessment

Metric 16:

Adequacy of Test Conditions

Medium

Photoperiod and temperature were reported, while other water quality conditions were
not.

Assessment was consistent among treatments and control.

Metric 17:
Metric 18:

Outcome Assessment Methodology

Consistency of Outcome
Assessment

Medium
High

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure

High
Medium

Nothing was reported to indicate differences in study groups based on environmental
factors.

No information in the study to indicate that differences were from animal attrition or
health outcomes.

Domain 7: Data Presentation and Analysis

Continued on next page .

Page 73 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 2953504 Table: 3 of 3

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Noyes, P. D., Haggard, D. E., Gonnerman, G. D., Tanguay, R. L. (2015). Advanced morphological - behavioral test platform reveals neurodevelopmental
defects in embryonic zebrafish exposed to comprehensive suite of halogenated and organophosphate flame retardants. Toxicological Sciences 145(1):177-
195.

Overall Duration: 4-10 days; Exposure Duration: 4-10 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ wild type (Tropical 5D); Embryo
Development/Growth
Tris(2-chloroethyl) phosphate (TCEP)

2953504

Domain

Metric

Rating

Comments

Metric 21:

Statistical Methods

High

"Data collection was undertaken using a custom barcoding and tracking system (Ze-
brafish Acquisition and Analysis Program) to facilitate reliable management of the large
amounts of data collected. Statistical analyses were performed using R code with test-
ing methodologies used by Truong et al. (2014) to evaluate developmental toxicity of
chemicals under the ToxCast program(RCoreTeam, 2014; Truong et al., 2014). Briefly,
a binomial testwas performed that calculated lowest effect levels (LELs) foreach end-
point to identify incidences that exceeded a significantthreshold above controls. This test
was preferable to a logisticregression as it accounted for the observed nonmonotonicity
offlame retardant toxicity."This reference is HERO ID 8591199

Metric 22:

Reporting of Data

High

All counts of data per treatment and control group are represented in the supplemental
data for each compound and time point (24 and 120 hpf).

Metric 23:

Explanation of Unexpected Outcomes

High

No unexpected outcomes were reported by the authors.

Additional Comments: This form is for developmental assessment at 120 hpf for TCEP. The specific data and statistical significance for each compound are located within the
	supplemental PDF.	

Overall Quality Determination

High

Page 74 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469203 Table: 1 of 2

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Sun, L., Xu, W., Peng, T.,ao, Chen, H., Ren, L.,in, Tan, H., Xiao, D.,an, Qian, H., Fu, Z. (2016). Developmental exposure of zebrafish larvae to
organophosphate flame retardants causes neurotoxicity. Neurotoxicology and Teratology 5516-22.

Overall Duration: 4-10 days; Exposure Duration: 4-10 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ Embryo
Behavioral

Tris(2-chloroethyl) phosphate (TCEP)

5469203

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

TCEP (CAS: 115-96-8; purity: 97% ).

Metric 2:

Test Substance Source

Low

Purchased from Sigma-Aldrich. The test substance identity was not analytically verified

by the performing laboratory.

Metric 3:

Test Substance Purity

High

97% purity reported.

Domain 2: Test Design

Metric 4:

Negative Controls

High

Solvent controls were used but solvent concentration in treatments was not reported.

Metric 5:

Negative Control Response

Low

Survival response of controls not reported. "No treatment-related effects were found in

the numbers of dead or malformed larvae for any chemical tested."

Metric 6:

Randomized Allocation

Medium

"The embryos (b2 h post fertilization (hpf)) were randomly transferred into individual

wells of 96-well plates (Corning, NY, USA) containing 100 "L of chemical solution."

Domain 3: Exposure Characterization

Metric 7:

Experimental System/Test Media

Medium

Test vessels were not covered.

Preparation

Metric 8:

Consistency of Exposure

High

Exposures were administered consistently.

Administration

Metric 9:

Measurement of Test Substance

Medium

Concentrations were not measured, but no reason to believe actual concentrations dis-

Concentration

similar from nominal.

Metric 10:

Exposure Duration and Frequency

High

Duration and frequency were appropriate.

Metric 11:

Number of Exposure Groups/

High

Number and spacing of groups was sufficient.

Spacing of Exposure Levels

Metric 12:

Testing at or Below Solubility Limit

High

Concentrations were below solubility values.

Domain 4: Test Organism

Metric 13:

Test Organism Characteristics

High

"The zebrafish (Danio rerio) originated from the Institute of Hydrobiology of the Chi-

nese Academy of Science (Wuhan, China). The fish maintenances were conducted ac-

cording to the method of Westerfield (2000) with minor modification (Sun et al., 2010)"

Metric 14:

Acclimatization and Pretreatment

High

Control and treatment organisms treated similarly.

Conditions

Metric 15:

Number of Organisms and

Medium

"Twenty embryos in one plate were used for one replicate, with triplicate plates for each

Replicates per Group

treatment."

Continued on next page ...

Page 75 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469203 Table: 1 of 2

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Sun, L., Xu, W., Peng, T.,ao, Chen, H., Ren, L.,in, Tan, H., Xiao, D.,an, Qian, H., Fu, Z. (2016). Developmental exposure of zebrafish larvae to
organophosphate flame retardants causes neurotoxicity. Neurotoxicology and Teratology 5516-22.

Overall Duration: 4-10 days; Exposure Duration: 4-10 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ Embryo
Behavioral

Tris(2-chloroethyl) phosphate (TCEP)

5469203

Domain

Metric

Rating

Comments

Domain 5: Outcome Assessment

Metric 16: Adequacy of Test Conditions

Metric 17: Outcome Assessment Methodology

Metric 18: Consistency of Outcome
	Assessment.	

Medium No details of test conditions were reported, but followed procedures from published

peer-reviewed studies. "The fishmaintenances were conducted according to the method
of Westerfield (2000) with minor modification (Sun et al., 2010)."

High Methods were reported for the outcomes of interest, "...the locomotor activity was mea-
sured by the Zebralab Video-track system..."

High No inconsistencies were noted.

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure

Low Environmental conditions and attrition were not reported.
Medium No information to suggest differences among groups

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods

Metric 22: Reporting of Data

Metric 23: Explanation of Unexpected Outcomes

High

High
High

Statistical methods clearly described. "Data were checked for normality and homo-
geneity of variance before conducting statistical comparison. As the assumptions were
met,the data were subjected to one-way analysis of variance (ANOVA)followed by Dun-
nett's post hoc test"

Data was reported in a reasonably clear manner.

No unexpected outcomes were reported

Additional Comments: This evaluation is for the behavioral outcome measured in zebrafish larvae following exposure to TCEP.

Overall Quality Determination

High

Page 76 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469203 Table: 2 of 2

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Overall Duration: 4-10 days; Exposure Duration: 4-10 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)
Vertebrate; Fish; Danio rerio\ Embryo

Mechanistic-Biomarkers (exposure and effect)-Cell signaling/function-Neurotoxicology

Tris(2-chloroethyl) phosphate (TCEP)

5469203

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

TCEP (CAS: 115-96-8; purity: 97% ).

Metric 2:

Test Substance Source

Low

Purchased from Sigma-Aldrich but the test substance identity was NOT analytically

verified by the performing laboratory.

Metric 3:

Test Substance Purity

High

97% purity reported.

Domain 2: Test Design

Metric 4:

Negative Controls

High

Solvent controls were used but solvent concentration in treatments was not reported.

Metric 5:

Negative Control Response

Low

Survival response of controls not reported. "No treatment-related effects were found in

the numbers of dead or malformed larvae for any chemical tested."

Metric 6:

Randomized Allocation

Medium

"The embryos (b2 h post fertilization (hpf)) were randomly transferred into individual

wells of 96-well plates (Corning, NY, USA) containing 100 "L of chemical solution."

Domain 3: Exposure Characterization

Metric 7:

Experimental System/Test Media

Medium

Test vessels were not covered.

Preparation

Metric 8:

Consistency of Exposure

High

Exposures were administered consistently.

Administration

Metric 9:

Measurement of Test Substance

Medium

Concentrations were not measured, but no reason to believe actual concentrations dis-

Concentration

similar from nominal.

Metric 10:

Exposure Duration and Frequency

High

Duration and frequency were appropriate.

Metric 11:

Number of Exposure Groups/

High

Number and spacing of groups was sufficient.

Spacing of Exposure Levels

Metric 12:

Testing at or Below Solubility Limit

High

Concentrations were below solubility values.

Domain 4: Test Organism

Metric 13:

Test Organism Characteristics

High

"The zebrafish (Danio rerio) originated from the Institute of Hydrobiology of the Chi-

nese Academy of Science (Wuhan, China). The fish maintenances were conducted ac-

cording to the method of Westerfield (2000) with minor modification (Sun et al., 2010)"

Metric 14:

Acclimatization and Pretreatment

High

Control and treatment organisms treated similarly.

Conditions

Metric 15:

Number of Organisms and

Medium

"Twenty embryos in one plate were used for one replicate, with triplicate plates for each

Replicates per Group

treatment."

Domain 5: Outcome Assessment

Continued on next page ...

Page 77 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469203 Table: 2 of 2

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Overall Duration: 4-10 days; Exposure Duration: 4-10 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)
Vertebrate; Fish; Danio rerio\ Embryo

Mechanistic-Biomarkers (exposure and effect)-Cell signaling/function-Neurotoxicology

Tris(2-chloroethyl) phosphate (TCEP)

5469203

Domain

Metric

Rating

Comments

Metric 16:

Adequacy of Test Conditions

Medium

No details of test conditions were reported, but followed procedures from published

peer-reviewed studies. "The fishmaintenances were conducted according to the method

of Westerfield (2000) with minor modification (Sun et al., 2010)."

Metric 17:

Outcome Assessment Methodology

High

Outcome assessment methodologies for AChE activity measurements and gene tran-

scription were provided in detail.

Metric 18:

Consistency of Outcome

High

No inconsistencies were noted.

Assessment

Domain 6: Confounding / Variable Control

Metric 19:

Confounding Variables in Test

Low

Environmental conditions and attrition were not reported.

Design and Procedures

Metric 20:

Outcomes Unrelated to Exposure

Medium

No information to suggest differences among groups

Domain 7: Data Presentation and Analysis

Metric 21:

Statistical Methods

High

Statistical methods clearly described. "Data were checked for normality and homo-

geneity of variance before conducting statistical comparison. As the assumptions were

met,the data were subjected to one-way analysis of variance (ANOVA)followed by Dun-

nett's post hoc test"

Metric 22:

Reporting of Data

High

Data was reported in a reasonably clear manner.

Metric 23:

Explanation of Unexpected Outcomes

High

No unexpected outcomes were reported

Additional Comments: This evaluation form is for the mechanistic data (AChE activity and gene transcription) in the reference.

Overall Quality Determination

High

Page 78 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469243 Table: 1 of 5

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Wu, Y., Su, G., Tang, S., Liu, W., Ma, Z., Zheng, X., Liu, H., Yu, H. (2017). The combination of in silico and in vivo approaches for the investigation of
disrupting effects of tris (2-chloroethyl) phosphate (TCEP) toward core receptors of zebraflsh. Chemosphere 168:122-130.

Overall Duration: 4-10 days; Exposure Duration: 4-10 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ AB strain; Embryo
Development/Growth
Tris(2-chloroethyl) phosphate (TCEP)

5469243

Domain

Metric

Rating

Comments

Domain 1: Test Substance







Metric 1:

Test Substance Identity

High

TCEP was purchased from AccuStandard (100% , AccuStandardlnc., CT, USA).

Metric 2:

Test Substance Source

Low

percent purity was not analytically verified

Metric 3:

Test Substance Purity

High

reported as 100% purity

Domain 2: Test Design







Metric 4:

Negative Controls

High

vehicle control reported with 0.1% DMSO

Metric 5:

Negative Control Response

High

control mortality was <= 8.3%

Metric 6:

Randomized Allocation

Medium

embryos were randomly distributed

Domain 3: Exposure Characterization







Metric 7:

Experimental System/Test Media

High

documentation was adequate for this test system



Preparation





Metric 8:

Consistency of Exposure

High

no inconsistencies were noted



Administration





Metric 9:

Measurement of Test Substance

Medium

concentrations were measured but results were reported as log concentrations. "Based



Concentration



on the preliminary test, a gradient of nominal concentrationswas chosen (2.85, 28.5,







285, 14,250 and 28,500 mg/L, equalto 0.01, 0.1, 1, 50 and 100 mM, respectively)."

Metric 10:

Exposure Duration and Frequency

High

3 to 120-h post fertilization

Metric 11:

Number of Exposure Groups/

High

a wide range of concentrations was reported



Spacing of Exposure Levels





Metric 12:

Testing at or Below Solubility Limit

High

the use of DMSO should be adequate to ensure the highest concentrations were fully







dissolved

Domain 4: Test Organism







Metric 13:

Test Organism Characteristics

High

age and source of organisms seemed satisfactory, although genus species was not spec-







ified by study authorsage and source of organisms seemed satisfactory"Adult zebrafish







(4 months old, AB wild-type) were obtainedfrom the Institute of Hydrobiology, Chinese







Academy of Sciences"

Metric 14:

Acclimatization and Pretreatment

Medium

pretreatment was not well documented but it was adequate



Conditions





Metric 15:

Number of Organisms and

Medium

three replicates were used



Replicates per Group





Continued on next page ...

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469243 Table: 1 of 5

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Wu, Y., Su, G., Tang, S., Liu, W., Ma, Z., Zheng, X., Liu, H., Yu, H. (2017). The combination of in silico and in vivo approaches for the investigation of
disrupting effects of tris (2-chloroethyl) phosphate (TCEP) toward core receptors of zebrafish. Chemosphere 168:122-130.

Overall Duration: 4-10 days; Exposure Duration: 4-10 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ AB strain; Embryo
Development/Growth
Tris(2-chloroethyl) phosphate (TCEP)

5469243

Domain

Metric

Rating

Comments

Domain 5: Outcome Assessment

Metric 16:
Metric 17:
Metric 18:

Adequacy of Test Conditions
Outcome Assessment Methodology
Consistency of Outcome
Assessment

Medium
Low
High

minimal documentation of exposure water conditions
malformations were not well quantified

observations were made at multiple durations for all concentrations

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure

High
High

no variables were reported
no unrelated outcomes reported

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods

Metric 22: Reporting of Data

Metric 23: Explanation of Unexpected Outcomes

Low
Low
High

malformations were not statistically analyzed

data was reported in the form of typical images and in text but not quantified
no unexpected outcomes reported

Additional Comments: None

Overall Quality Determination

High

Page 80 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469243 Table: 2 of 5

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Wu, Y., Su, G., Tang, S., Liu, W., Ma, Z., Zheng, X., Liu, H., Yu, H. (2017). The combination of in silico and in vivo approaches for the investigation of
disrupting effects of tris (2-chloroethyl) phosphate (TCEP) toward core receptors of zebraflsh. Chemosphere 168:122-130.

Overall Duration: 4-10 days; Exposure Duration: 4-10 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ AB strain; Embryo
Mechanistic-Biomarkers (exposure and effect)-Cell signaling/function
Tris(2-chloroethyl) phosphate (TCEP)

5469243

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1:
Metric 2:
Metric 3:

Test Substance Identity
Test Substance Source
Test Substance Purity

High
Low
High

TCEP was purchased from AccuStandard (100% , AccuStandardlnc., CT, USA),
percent purity was not analytically verified

reported as 100% purity'TCEP was purchased from AccuStandard (100% , AccuStan-
dardlnc., CT, USA)."

Domain 2: Test Design

Metric 4:
Metric 5:
Metric 6:

Negative Controls
Negative Control Response
Randomized Allocation

High
High
Medium

vehicle control reported with 0.1% DMSO
control mortality was <= 8.3%
embryos were randomly distributed

Domain 3: Exposure Characterization
Metric 7:

Metric 8:

Metric 9:

Metric 10:
Metric 11:

Experimental System/Test Media
Preparation

Consistency of Exposure
Administration

Measurement of Test Substance
Concentration

Exposure Duration and Frequency
Number of Exposure Groups/
Spacing of Exposure Levels

High

High

Medium

High
High

documentation was adequate for this test system
no inconsistencies were noted

concentrations were measured but results were reported as log concentrations
3 to 120-h post fertilization

a wide range of concentrations was reported "Expression of genes associated with es-
trogen receptor in zebraflsh larvae (120hpf) exposure to 2.85, 28.5 and 285 mg TCEP/
L "

Metric 12:

Testing at or Below Solubility Limit

High

the use of DMSO should be adequate to ensure the highest concentrations were fully
dissolved

Domain 4: Test Organism

Metric 13:
Metric 14:

Metric 15:

Test Organism Characteristics
Acclimatization and Pretreatment
Conditions

Number of Organisms and
Replicates per Group

High
Medium

Medium

age and source of organisms seemed satisfactory
pretreatment was not well documented but it was adequate

three replicates were used

Domain 5: Outcome Assessment

Metric 16:

Adequacy of Test Conditions

Medium

minimal documentation of exposure water conditions

Continued on next page ...

Page 81 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469243 Table: 2 of 5

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

... continued from previous page

Wu, Y., Su, G., Tang, S., Liu, W., Ma, Z., Zheng, X., Liu, H., Yu, H. (2017). The combination of in silico and in vivo approaches for the investigation of
disrupting effects of tris (2-chloroethyl) phosphate (TCEP) toward core receptors of zebrafish. Chemosphere 168:122-130.

Overall Duration: 4-10 days; Exposure Duration: 4-10 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ AB strain; Embryo
Mechanistic-Biomarkers (exposure and effect)-Cell signaling/function
Tris(2-chloroethyl) phosphate (TCEP)

5469243

Domain

Metric

Rating

Comments

Metric 17:

Outcome Assessment Methodology

High

assessment of endocrine gene expression was adequateThere were some concerns, how-
ever, regarding the different responses among exposure concentrations. Three concentra-
tions of TCEP/L were asssessed, 2.85, 28.5, and 285 ug TCEP/L. Results for the vtg2,
pgr, ncoal, ncoa3, er2b, vtgl, and erl indicated a greater response for 2.85 and 285 ug
TCEP/L than the middle 28.5 concentration. The authors did not provide a rationale as
to why this may have occurred.

Metric 18:

Consistency of Outcome
Assessment.

High

no inconsistencies were reported

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure

High no confounding variables were reported
High no unrelated outcomes reported

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods

Metric 22: Reporting of Data

Metric 23: Explanation of Unexpected Outcomes

High

High
High

"Key receptor pathway analysis was conducted according to a previous study (Liu et
al., 2015). The resulting network genes (nodes) were colored by the Enhanced Graphics
application within Cytoscape v3.1.1 (Cytoscape consortium, San Diego, CA, USA)
according to the significant changes in gene expression in the respective treatments."
data was clearly reported graphically and in text
no unexpected outcomes reported

Additional Comments: Due higher responses seen at the highest 285 ug TCEP/L and lowest 2.85 ug TCEP/L concentrations compared to the middle 28.5 ug TCEP/L concentra-
	tions, it is not clear which concentration is responsible for the effect seen.	

Overall Quality Determination	High

Page 82 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469243 Table: 3 of 5

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Wu, Y., Su, G., Tang, S., Liu, W., Ma, Z., Zheng, X., Liu, H., Yu, H. (2017). The combination of in silico and in vivo approaches for the investigation of
disrupting effects of tris (2-chloroethyl) phosphate (TCEP) toward core receptors of zebraflsh. Chemosphere 168:122-130.

Overall Duration: 4-10 days; Exposure Duration: 4-10 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ AB strain; Embryo
Endocrine

Tris(2-chloroethyl) phosphate (TCEP)

5469243

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1:
Metric 2:
Metric 3:

Test Substance Identity
Test Substance Source
Test Substance Purity

High
Low
High

TCEP was purchased from AccuStandard (100% , AccuStandardlnc., CT, USA),
percent purity was not analytically verified

reported as 100% purity'TCEP was purchased from AccuStandard (100% , AccuStan-
dardlnc., CT, USA)."

Domain 2: Test Design

Metric 4:
Metric 5:
Metric 6:

Negative Controls
Negative Control Response
Randomized Allocation

High
High
Medium

vehicle control reported with 0.1% DMSO
control mortality was <= 8.3%
embryos were randomly distributed

Domain 3: Exposure Characterization
Metric 7:

Metric 8:

Metric 9:

Metric 10:
Metric 11:

Experimental System/Test Media
Preparation

Consistency of Exposure
Administration

Measurement of Test Substance
Concentration

Exposure Duration and Frequency
Number of Exposure Groups/
Spacing of Exposure Levels

High

High

Medium

High
High

documentation was adequate for this test system
no inconsistencies were noted

concentrations were measured but results were reported as log concentrations
3 to 120-h post fertilization

a wide range of concentrations was reported "Expression of genes associated with es-
trogen receptor in zebraflsh larvae (120hpf) exposure to 2.85, 28.5 and 285 mg TCEP/
L "

Metric 12:

Testing at or Below Solubility Limit

High

the use of DMSO should be adequate to ensure the highest concentrations were fully
dissolved

Domain 4: Test Organism

Metric 13:
Metric 14:

Metric 15:

Test Organism Characteristics
Acclimatization and Pretreatment
Conditions

Number of Organisms and
Replicates per Group

High
Medium

Medium

age and source of organisms seemed satisfactory
pretreatment was not well documented but it was adequate

three replicates were used

Domain 5: Outcome Assessment

Metric 16:

Adequacy of Test Conditions

Medium

minimal documentation of exposure water conditions

Continued on next page ...

Page 83 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469243 Table: 3 of 5

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Wu, Y., Su, G., Tang, S., Liu, W., Ma, Z., Zheng, X., Liu, H., Yu, H. (2017). The combination of in silico and in vivo approaches for the investigation of
disrupting effects of tris (2-chloroethyl) phosphate (TCEP) toward core receptors of zebrafish. Chemosphere 168:122-130.

Overall Duration: 4-10 days; Exposure Duration: 4-10 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ AB strain; Embryo
Endocrine

Tris(2-chloroethyl) phosphate (TCEP)

5469243

Domain

Metric

Rating

Comments

Metric 17:

Outcome Assessment Methodology

High

assessment of endocrine gene expression was adequateThere were some concerns, how-







ever, regarding the different responses among exposure concentrations. Three concentra-







tions of TCEP/L were asssessed, 2.85, 28.5, and 285 ug TCEP/L. Results for the vtg2,







pgr, ncoal, ncoa3, er2b, vtgl, and erl indicated a greater response for 2.85 and 285 ug







TCEP/L than the middle 28.5 concentration. The authors did not provide a rationale as







to why this may have occurred.

Metric 18:

Consistency of Outcome

High

no inconsistencies were reported



Assessment





Domain 6: Confounding / Variable Control





Metric 19:

Confounding Variables in Test

High

no confounding variables were reported



Design and Procedures





Metric 20:

Outcomes Unrelated to Exposure

High

no unrelated outcomes reported

Domain 7: Data Presentation and Analysis





Metric 21:

Statistical Methods

High

"Key receptor pathway analysis was conducted according to a previous study (Liu et







al., 2015). The resulting network genes (nodes) were colored by the Enhanced Graphics







application within Cytoscape v3.1.1 (Cytoscape consortium, San Diego, CA, USA)







according to the significant changes in gene expression in the respective treatments."

Metric 22:

Reporting of Data

High

data was clearly reported graphically and in text

Metric 23:

Explanation of Unexpected Outcomes

High

no unexpected outcomes reported

Additional Comments:

Due higher responses seen at the highest 285 ug TCEP/L and lowest 2.85 ug TCEP/L concentrations compared to the middle 28.5 ug TCEP/L concentra-
tions, it is not clear which concentration is responsible for the effect seen.	

Overall Quality Determination

High

Page 84 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469243 Table: 4 of 5

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Wu, Y., Su, G., Tang, S., Liu, W., Ma, Z., Zheng, X., Liu, H., Yu, H. (2017). The combination of in silico and in vivo approaches for the investigation of
disrupting effects of tris (2-chloroethyl) phosphate (TCEP) toward core receptors of zebraflsh. Chemosphere 168:122-130.

Overall Duration: 4-10 days; Exposure Duration: 4-10 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ AB strain; Embryo
Mortality

Tris(2-chloroethyl) phosphate (TCEP)

5469243

Domain

Metric

Rating

Comments

Domain 1: Test Substance







Metric 1:

Test Substance Identity

High

TCEP was purchased from AccuStandard (100% , AccuStandardlnc., CT, USA).

Metric 2:

Test Substance Source

Low

percent purity was not analytically verified

Metric 3:

Test Substance Purity

High

reported as 100% purity

Domain 2: Test Design







Metric 4:

Negative Controls

High

vehicle control reported with 0.1% DMSO

Metric 5:

Negative Control Response

High

control mortality was <= 8.3% "The morality rate of each concentration (2.85, 28.5and







28,500 mg TCEP/L) did not change rapidly (the change ofmortality rate was no more







than 12.5% ) from 24 to 120 hpf"

Metric 6:

Randomized Allocation

Medium

embryos were randomly distributed

Domain 3: Exposure Characterization







Metric 7:

Experimental System/Test Media

High

documentation was adequate for this test system



Preparation





Metric 8:

Consistency of Exposure

High

no inconsistencies were noted



Administration





Metric 9:

Measurement of Test Substance

Medium

concentrations were measured but results were reported as log concentrations



Concentration





Metric 10:

Exposure Duration and Frequency

High

3 to 120-h post fertilization

Metric 11:

Number of Exposure Groups/

High

a wide range of concentrations was reported



Spacing of Exposure Levels





Metric 12:

Testing at or Below Solubility Limit

High

the use of DMSO should be adequate to ensure the highest concentrations were fully

dissolved

Domain 4: Test Organism

Metric 13: Test Organism Characteristics	High age and source of organisms seemed satisfactory"Adult zebraflsh (4 months old, AB

wild-type) were obtainedfrom the Institute of Hydrobiology, Chinese Academy of Sci-
ences"

Metric 14: Acclimatization and Pretreatment	Medium pretreatment was not well documented but it was adequate

Conditions

Metric 15: Number of Organisms and Medium three replicates were used
	Replicates per Group	

Domain 5: Outcome Assessment

Metric 16:

Adequacy of Test Conditions

Medium minimal documentation of exposure water conditions

Continued on next page ...

Page 85 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469243 Table: 4 of 5

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Wu, Y., Su, G., Tang, S., Liu, W., Ma, Z., Zheng, X., Liu, H., Yu, H. (2017). The combination of in silico and in vivo approaches for the investigation of
disrupting effects of tris (2-chloroethyl) phosphate (TCEP) toward core receptors of zebrafish. Chemosphere 168:122-130.

Overall Duration: 4-10 days; Exposure Duration: 4-10 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ AB strain; Embryo
Mortality

Tris(2-chloroethyl) phosphate (TCEP)

5469243

Domain

Metric

Rating

Comments

Metric 17:
Metric 18:

Outcome Assessment Methodology
Consistency of Outcome
Assessment

High
High

both percent mortality and log LC50 values were reported at multiple durations
observations were made at multiple durations for all concentrations

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure

High
High

no variables were reported
no unrelated outcomes reported

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods

Metric 22: Reporting of Data

Metric 23: Explanation of Unexpected Outcomes

High
High
High

both percent mortality and Log LC50 values reported at various durations
data was reported graphically and in text
no unexpected outcomes reported

Additional Comments: None

Overall Quality Determination

High

Page 86 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469243 Table: 5 of 5

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Wu, Y., Su, G., Tang, S., Liu, W., Ma, Z., Zheng, X., Liu, H., Yu, H. (2017). The combination of in silico and in vivo approaches for the investigation of
disrupting effects of tris (2-chloroethyl) phosphate (TCEP) toward core receptors of zebraflsh. Chemosphere 168:122-130.

Overall Duration: 4-10 days; Exposure Duration: 4-10 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ AB strain; Embryo
ADME (biotransformation)

Tris(2-chloroethyl) phosphate (TCEP)

5469243

Domain

Metric

Rating

Comments

Domain 1: Test Substance







Metric 1:

Test Substance Identity

High

TCEP was purchased from AccuStandard (100% , AccuStandardlnc., CT, USA).

Metric 2:

Test Substance Source

Low

percent purity was not analytically verified

Metric 3:

Test Substance Purity

High

reported as 100% purity

Domain 2: Test Design







Metric 4:

Negative Controls

High

vehicle control reported with 0.1% DMSO

Metric 5:

Negative Control Response

Low

control response was not reported

Metric 6:

Randomized Allocation

Medium

embryos were randomly distributed

Domain 3: Exposure Characterization







Metric 7:

Experimental System/Test Media

High

documentation was adequate for this test system



Preparation





Metric 8:

Consistency of Exposure

High

no inconsistencies were noted by the study authors



Administration





Metric 9:

Measurement of Test Substance

High

concentrations were measured but actual values were not reported except as the Log



Concentration



value of the mean-measured concentrations

Metric 10:

Exposure Duration and Frequency

High

3 to 120-h post fertilization

Metric 11:

Number of Exposure Groups/

High

a wide range of concentrations was reported



Spacing of Exposure Levels





Metric 12:

Testing at or Below Solubility Limit

High

the use of DMSO should be adequate to ensure the highest concentrations were fully







dissolved

Domain 4: Test Organism







Metric 13:

Test Organism Characteristics

High

age and source of organisms seemed satisfactory, although genus species was not spec-







ified by study authorsage and source of organisms seemed satisfactory"Adult zebrafish







(4 months old, AB wild-type) were obtainedfrom the Institute of Hydrobiology, Chinese







Academy of Sciences"

Metric 14:

Acclimatization and Pretreatment

Low

pretreatment was not well documented but it was adequate



Conditions





Metric 15:

Number of Organisms and

Medium

number of organisms and replicates (3) were adequate



Replicates per Group





Domain 5: Outcome Assessment

Continued on next page ...

Page 87 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469243 Table: 5 of 5

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Wu, Y., Su, G., Tang, S., Liu, W., Ma, Z., Zheng, X., Liu, H., Yu, H. (2017). The combination of in silico and in vivo approaches for the investigation of
disrupting effects of tris (2-chloroethyl) phosphate (TCEP) toward core receptors of zebrafish. Chemosphere 168:122-130.

Overall Duration: 4-10 days; Exposure Duration: 4-10 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Danio rerio\ AB strain; Embryo
ADME (biotransformation)

Tris(2-chloroethyl) phosphate (TCEP)

5469243

Domain

Metric

Rating

Comments

Metric 16:

Adequacy of Test Conditions

Low

minimal documentation of exposure water conditions "Moreover, the morality rate of







each concentration (2.85, 28.5and 28,500 mg TCEP/L) did not change rapidly (the







change ofmortality rate was no more than 12.5% ) from 24 to 120 hpf (seeSupplemen-







tary Table S2), indicating the amount of chemicalentering the zebrafish did not change







greatly with increasing ofexposure concentrations, consistent with the relatively low log







Kow(1.44)." The second half of that statement seems to indicate that, contrary to what







the study authors have concluded (a relationship between bioaccumulation and TCEP







exposure), increasing the TCEP concentrations had no effect on bioaccumulation by







zebrafish.

Metric 17:

Outcome Assessment Methodology

Medium

The bioaccumulation potency of TCEP was calculated based on the ratios of concentra-







tions between zebrafish larvae and exposure solutions.

Metric 18:

Consistency of Outcome

Medium

control measurements were not well documented, other sources were cited and were



Assessment



reviewed to determine assessment methods

Domain 6: Confounding / Variable Control





Metric 19:

Confounding Variables in Test

High

no variables were reported



Design and Procedures





Metric 20:

Outcomes Unrelated to Exposure

High

no unexpected outcomes reported

Domain 7: Data Presentation and Analysis





Metric 21:

Statistical Methods

High

a linear regression equation was established and there was little statistical difference







between nominal and measured test concentrations

Metric 22:

Reporting of Data

Medium

actual measured test concentrations that accumulation was based on, was only available







in log values

Metric 23:

Explanation of Unexpected Outcomes

High

no unexpected outcomes reported, expected outcomes had explanations provided

Additional Comments: None

Overall Quality Determination

High

Page 88 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 4292102 Table: 1 of 5

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Sun, L., Tan, H., Peng, T., Wang, S., Xu, W., Qian, H., Jin, Y., Fu, Z. (2016). Developmental neurotoxicity of organophosphate flame retardants in early
life stages of Japanese medaka (Oryzias latipes). Environmental Toxicology and Chemistry 35(12):2931-2940.

Overall Duration: 0 - 4 days (0-96h); Exposure Duration: 0 - 4 days (0-96h)

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Oryzias latipes\ d-rR strain; Larvae
Behavioral

Tris(2-chloroethyl) phosphate (TCEP)

4292102

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1: Test Substance Identity
Metric 2: Test Substance Source
Metric 3: Test Substance Purity

High The test substance was identified as tris (2-chloroethyl) phosphate (TCEP; CAS no.

115-96-8). Chemical structure was given in table 1.

Low Purchased from Sigma-Aldrich. The test substance identity was not analytically verified

by the performing laboratory.

High Percent purity was reported as 97% .

Domain 2: Test Design

Metric 4: Negative Controls
Metric 5: Negative Control Response

Metric 6: Randomized Allocation

High A control group containing 0.01% DMSO was included.

High Control group response for behavior assay were reported in the text and in figures 1 and
2.

Low Researchers did not report how organisms were allocated to study groups. Non-
deformed larvae were selected for behavior assay

Domain 3: Exposure Characterization

Metric 7:

Experimental System/Test Media

High

Preparation

Metric 8:

Consistency of Exposure

High

Administration

Metric 9:

Measurement of Test Substance

Low

Concentration

Metric 10:

Exposure Duration and Frequency

High

Metric 11:

Number of Exposure Groups/

High

Spacing of Exposure Levels

Metric 12:

Testing at or Below Solubility Limit

High

Static system, test solution renewed every 24 hrs.

Exposures were consistent among all test groups.

Exposure concentrations were not measured.

Exposure duration of 96 hours was appropriate for test.

The number of exposure groups and spacing of exposure levels were adequate to address
the purpose of the study. 4 concentrations and a control (5 mg/L, 25 mg/L, 125 mg/L,
625 mg/L) w were used.

Exposure concentrations were at or below the solubility limit. The solvent concentration
was appropriate.

Domain 4: Test Organism

Metric 13: Test Organism Characteristics

Metric 14: Acclimatization and Pretreatment
Conditions

High Japanese medaka (d-rR strain) originated from the Laboratory of Freshwater Fish at the

Bioscience Center of Nagoya University.

Low The study did not report whether test organisms were acclimatized and/or whether pre-
treatment conditions were the same for control and exposed groups.

Continued on next page ...

Page 89 of 170

-------
Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 4292102 Table: 1 of 5

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Sun, L., Tan, H., Peng, T., Wang, S., Xu, W., Qian, H., Jin, Y., Fu, Z. (2016). Developmental neurotoxicity of organophosphate flame retardants in early
life stages of Japanese medaka (Oryzias latipes). Environmental Toxicology and Chemistry 35(12):2931-2940.

Overall Duration: 0 - 4 days (0-96h); Exposure Duration: 0 - 4 days (0-96h)

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Oryzias latipes\ d-rR strain; Larvae
Behavioral

Tris(2-chloroethyl) phosphate (TCEP)

4292102

Domain

Metric

Rating

Comments

Metric 15:

Number of Organisms and
Replicates per Group

Medium

20 test organisms with 3 replicates per treatment.

Domain 5: Outcome Assessment

Metric 16:

Metric 17:
Metric 18:

Adequacy of Test Conditions
Outcome Assessment Methodology

Consistency of Outcome
Assessment

Low
High

High

Housing and environmental conditions were adequate during incubation period. How-
ever, environmental conditions were not reported for test conditions.

Outcome assessment methodology (locomotor behavior assay) was appropriate and re-
ported the intended outcomes of interest. End points were changes in the relative swim-
ming speed during 30 min of visible light and during the dark-light-dark photoperiod
stimulation test

Outcome assessments were consistent across study groups.

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure

Low
Medium

Environmental conditions during test were not reported.

There was no information in the study to suggest differences among groups in animal
attrition or health outcomes unrelated to exposure (e.g., infection) that could influence

the outcome assessment.

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods	High Appropriate statistics were used after data met assumptions of the tests.

Metric 22: Reporting of Data	High Data for exposure-related findings (changes in the relative swimming speed during 30

min of visible light and during the dark-light-dark photoperiod stimulation test; figures 1
and 2) were presented for each treatment and control group
Metric 23: Explanation of Unexpected Outcomes High No unexpected outcomes were reported.

Additional Comments: This evaluation form is relevant to the behavioral outcome (changes in the relative swimming speed during 30 min of visible light and during the dark-
	light-dark photoperiod stimulation test) in medaka larvae following exposure to TCEP.	

Overall Quality Determination	High

Page 90 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 4292102 Table: 2 of 5

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Overall Duration: 0 - 4 days (0-96h); Exposure Duration: 0 - 4 days (0-96h)

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Oryzias latipes\ d-rR strain; Larvae
Mechanistic-Biomarkers (exposure and effect)-Cell signaling/function
Tris(2-chloroethyl) phosphate (TCEP)

4292102

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

The test substance was identified as tris (2-chloroethyl) phosphate (TCEP; CAS no.

115-96-8). Chemical structure was given in table 1.

Metric 2:

Test Substance Source

Low

Purchased from Sigma-Aldrich. The test substance identity was not analytically verified

by the performing laboratory.

Metric 3:

Test Substance Purity

High

Percent purity was reported as 97%

Domain 2: Test Design

Metric 4:

Negative Controls

High

A control group containing 0.01% DMSO was included.

Metric 5:

Negative Control Response

High

Control group response for AChE and mRNA expression were reported in the text and in

Figures 3 and 4. Control responses were adequate.

Metric 6:

Randomized Allocation

Low

Researchers did not report how organisms were allocated to study groups.

Domain 3: Exposure Characterization

Metric 7:

Experimental System/Test Media

High

Static system, test solution renewed every 24 hrs.

Preparation

Metric 8:

Consistency of Exposure

High

Exposures were consistent among all test groups.

Administration

Metric 9:

Measurement of Test Substance

Low

Exposure concentrations were not measured.

Concentration

Metric 10:

Exposure Duration and Frequency

High

Exposure duration (96 hours) appropriate for test.

Metric 11:

Number of Exposure Groups/

High

The number of exposure groups and spacing of exposure levels were adequate to address

Spacing of Exposure Levels

the purpose of the study. 4 concentrations and a control (5 mg/L, 25 mg/L, 125 mg/L,

and 625 mg/L).

Metric 12:

Testing at or Below Solubility Limit

High

Exposure concentrations were at or below the solubility limit. The solvent concentration

was appropriate.

Domain 4: Test Organism

Metric 13:

Test Organism Characteristics

High

Japanese medaka (d-rR strain) originated from the Laboratory of Freshwater Fish at the

Bioscience Center of Nagoya University.

Metric 14:

Acclimatization and Pretreatment

Low

The study did not report whether test organisms were acclimatized and/or whether pre-

Conditions

treatment conditions were the same for control and exposed groups.

Metric 15:

Number of Organisms and

Medium

For gene transcription analysis, 15 larvae from each replicate (from the behavior assay)

Replicates per Group

were pooled into 1 sample. For AChE activity measurements, 30 larvae/beaker were

sampled and homogenized after the exposure period, and 3 replicates of each concentra-

tion were used.

Continued on next page ...

Page 91 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 4292102 Table: 2 of 5

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

... continued from previous page

Overall Duration: 0 - 4 days (0-96h); Exposure Duration: 0 - 4 days (0-96h)

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Oryzias latipes\ d-rR strain; Larvae
Mechanistic-Biomarkers (exposure and effect)-Cell signaling/function
Tris(2-chloroethyl) phosphate (TCEP)

4292102

Domain

Metric

Rating

Comments

Domain 5: Outcome Assessment

Metric 16:

Metric 17:

Metric 18:

Adequacy of Test Conditions

Outcome Assessment Methodology

Consistency of Outcome
Assessment.

Low Housing and environmental conditions were adequate during incubation period. How-
ever, environmental conditions were not reported for test conditions.

High Outcome assessment methodologies (for acetylcholinesterase activity and gene tran-
scription analysis) was appropriate and reported the intended outcomes of interest.
High Outcome assessments were consistent across study groups.

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure

Low Environmental conditions during test were not reported.

Medium There was no information in the study to suggest differences among groups in animal
attrition or health outcomes unrelated to exposure (e.g., infection) that could influence
the outcome assessment.

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods High Appropriate statistics were used after data met assumptions of the tests.

Metric 22: Reporting of Data High Data for exposure-related findings for AChE activity and mRNA level (Figures 3 and

4) were presented for each treatment and control group and were adequate to determine
values for the endpoints of interest.

Metric 23: Explanation of Unexpected Outcomes High No unexpected outcomes were reported.

Additional Comments: This evaluation form is relevant to the mechanistic endpoints (AChE activity and changes in the transcription of genes related to the nervous system) in
medaka larvae following exposure to TPP

Overall Quality Determination High

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Tris(2-chloroethyl) phosphate (TCEP) Environmental Hazard Evaluation HERO ID: 4292102 Table: 3 of 5

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Overall Duration: 11-21 days; Exposure Duration: 11-21 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Oryzias latipes\ d-rR strain; Embryo
Cardiovascular

Tris(2-chloroethyl) phosphate (TCEP)

4292102

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1: Test Substance Identity
Metric 2: Test Substance Source
Metric 3: Test Substance Purity

High The test substance was identified as tris (2-chloroethyl) phosphate (TCEP; CAS no.

115-96-8). Chemical structure was given in table 1.

Low Purchased from Sigma-Aldrich. The test substance identity was not analytically verified

by the performing laboratory.

High Percent purity reported as 97% .

Domain 2: Test Design

Metric 4: Negative Controls High A control group containing 0.01% DMSO was included.

Metric 5: Negative Control Response High Control group response (table 2) was adequate.

Metric 6: Randomized Allocation Medium Organisms were randomly allocated for treatments.

Domain 3: Exposure Characterization

Metric

Experimental System/Test Media
Preparation

High

Static system, test solution renewed every 24 hrs.

Metric

Consistency of Exposure
Administration

High

Exposures were consistent among all test groups.

Metric

Measurement of Test Substance
Concentration

Low

Exposure concentrations were not measured.

Metric

10:

Exposure Duration and Frequency

High

Exposure duration (14 days) appropriate for test. "During the exposure period, embryos
went through the major embryonic stages (blastula, gastrula, neurula, and organogene-
sis), hatching and proceeding into the larval stage."

Metric

11:

Number of Exposure Groups/

High

Number of exposure groups and dose spacing were appropriate for test. 4 concentra-

Spacing of Exposure Levels

tions and a control (5 mg/L, 25 mg/L, 125 mg/L, and 625 mg/L). Concentrations were

determined via a prior range finding test (data not shown).

Metric

12:

Testing at or Below Solubility Limit

High

Exposure concentrations were at or below the solubility limit. The solvent concentration

was appropriate.

Domain 4: Test Organism

Metric

13:

Test Organism Characteristics

High

Japanese medaka (d-rR strain) originated from the Laboratory of Freshwater Fish at the
Bioscience Center of Nagoya University.

Metric

14:

Acclimatization and Pretreatment
Conditions

Low

The study did not report whether test organisms were acclimatized and/or whether pre-
treatment conditions were the same for control and exposed groups.

Metric

15:

Number of Organisms and
Replicates per Group

Medium

20 test organisms with 3 replicates per treatment.

Continued on next page ...

Page 93 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 4292102 Table: 3 of 5

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Overall Duration: 11-21 days; Exposure Duration: 11-21 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Oryzias latipes\ d-rR strain; Embryo
Cardiovascular

Tris(2-chloroethyl) phosphate (TCEP)

4292102

Domain

Metric

Rating

Comments

Domain 5: Outcome Assessment

Metric 16:

Metric 17:

Metric 18:

Adequacy of Test Conditions

Outcome Assessment Methodology

Consistency of Outcome
Assessment.

Low Housing and environmental conditions were adequate during incubation period. How-
ever, environmental conditions were not reported for test conditions.

High Assessment methods for body length, hatch % , incubation time, deformity % and heart

rate were reported.

High Outcome assessments were consistent across study groups.

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure

Low The study did not provide enough information to allow a comparison of environmental
conditions or other non-treatment-related factors across study groups.

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods
Metric 22: Reporting of Data

Metric 23: Explanation of Unexpected Outcomes

High
High

High

Appropriate statistics were used after data met assumptions of the tests.

Data for exposure-related findings were presented for each treatment and control group
and were adequate to determine values for hatchability, time to hatching, gross abnor-
mality rate, heart rate and body length.

No unexpected outcomes were reported.

Additional Comments: This evaluation form is relevant to cardiovascular outcome (heart rate) determined in medaka embryos following exposure to TCEP.

Overall Quality Determination

High

Page 94 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 4292102 Table: 4 of 5

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Overall Duration: 11-21 days; Exposure Duration: 11-21 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Oryzias latipes\ d-rR strain; Embryo

Reproductive/Teratogenic

Tris(2-chloroethyl) phosphate (TCEP)

4292102

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

The test substance was identified as tris (2-chloroethyl) phosphate (TCEP; CAS no.

115-96-8). Chemical structure was given in table 1.

Metric 2:

Test Substance Source

Low

Purchased from Sigma-Aldrich. The test substance identity was not analytically verified

by the performing laboratory.

Metric 3:

Test Substance Purity

High

Percent purity reported as 97% .

Domain 2: Test Design

Metric 4:

Negative Controls

High

A control group containing 0.01% DMSO was included.

Metric 5:

Negative Control Response

High

Control group response (table 2) was adequate.

Metric 6:

Randomized Allocation

Medium

Organisms were randomly allocated for treatments.

Domain 3: Exposure Characterization

Metric 7:

Experimental System/Test Media

High

Static system, test solution renewed every 24 hrs.

Preparation

Metric 8:

Consistency of Exposure

High

Exposures were consistent among all test groups.

Administration

Metric 9:

Measurement of Test Substance

Low

Exposure concentrations were not measured.

Concentration

Metric 10:

Exposure Duration and Frequency

High

Exposure duration (14 days) appropriate for test. "During the exposure period, embryos

went through the major embryonic stages (blastula, gastrula, neurula, and organogene-

sis), hatching and proceeding into the larval stage."

Metric 11:

Number of Exposure Groups/

High

The number of exposure groups and spacing of exposure levels were adequate to address

Spacing of Exposure Levels

the purpose of the study. 4 concentrations and a control (5 mg/L, 25 mg/L, 125 mg/L,

and 625 mg/L). Concentrations were determined via a prior range finding test (data not

shown).

Metric 12:

Testing at or Below Solubility Limit

High

Exposure concentrations were at or below the solubility limit. The solvent concentration

was appropriate.

Domain 4: Test Organism

Metric 13:

Test Organism Characteristics

High

Japanese medaka (d-rR strain) originated from the Laboratory of Freshwater Fish at the

Bioscience Center of Nagoya University.

Metric 14:

Acclimatization and Pretreatment

Low

The study did not report whether test organisms were acclimatized and/or whether pre-

Conditions

treatment conditions were the same for control and exposed groups.

Metric 15:

Number of Organisms and

Medium

20 test organisms with 3 replicates per treatment.

Replicates per Group

Continued on next page ...

Page 95 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 4292102 Table: 4 of 5

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Overall Duration: 11-21 days; Exposure Duration: 11-21 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Oryzias latipes\ d-rR strain; Embryo

Reproductive/Teratogenic

Tris(2-chloroethyl) phosphate (TCEP)

4292102

Domain

Metric

Rating

Comments

Domain 5: Outcome Assessment

Metric 16:

Metric 17:

Metric 18:

Adequacy of Test Conditions

Outcome Assessment Methodology

Consistency of Outcome
Assessment.

Low Housing and environmental conditions were adequate during incubation period. How-
ever, environmental conditions were not reported for test conditions.

High Outcome assessment methods for body length, hatch % , incubation time, deformity %

and heart rate were reported.

High Outcome assessments were consistent across study groups.

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure

Low Environmental conditions during test were not reported.

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods
Metric 22: Reporting of Data

Metric 23: Explanation of Unexpected Outcomes

High
High

High

Appropriate statistics were used after data met assumptions of the tests.

No unexpected outcomes were reported.

Additional Comments: This evaluation form is relevant to reproductive outcomes (percent hatchability and time to hatch) determined in medaka embryos following exposure to
TCEP

Overall Quality Determination

High

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December 2023

Tris(2-chloroethyl) phosphate (TCEP) Environmental Hazard Evaluation HERO ID: 4292102 Table: 5 of 5

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Overall Duration: 11-21 days; Exposure Duration: 11-21 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Oryzias latipes\ d-rR strain; Embryo
Development/Growth
Tris(2-chloroethyl) phosphate (TCEP)

4292102

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1: Test Substance Identity
Metric 2: Test Substance Source
Metric 3: Test Substance Purity

High The test substance was identified as tris (2-chloroethyl) phosphate (TCEP; CAS no.

115-96-8). Chemical structure was given in table 1.

Low Purchased from Sigma-Aldrich. The test substance identity was not analytically verified

by the performing laboratory.

High Percent purity reported as 97% .

Domain 2: Test Design

Metric 4: Negative Controls High A control group containing 0.01% DMSO was included.

Metric 5: Negative Control Response High Control group response (table 2) was adequate.

Metric 6: Randomized Allocation Medium Organisms were randomly allocated for treatments.

Domain 3: Exposure Characterization

Metric

Experimental System/Test Media
Preparation

High

Static system, test solution renewed every 24 hrs.

Metric

Consistency of Exposure
Administration

High

Exposures were consistent among all test groups.

Metric

Measurement of Test Substance
Concentration

Low

Exposure concentrations were not measured.

Metric

10:

Exposure Duration and Frequency

High

Metric

11:

Number of Exposure Groups/

High

Number of exposure groups and dose spacing were appropriate for test. 4 concentra-

Spacing of Exposure Levels

tions and a control (5 mg/L, 25 mg/L, 125 mg/L, and 625 mg/L). Concentrations were

determined via a prior range finding test (data not shown).

Metric

12:

Testing at or Below Solubility Limit

High

Exposure concentrations were at or below the solubility limit. The solvent concentration

was appropriate.

Domain 4: Test Organism

Metric

13:

Test Organism Characteristics

High

Japanese medaka (d-rR strain) originated from the Laboratory of Freshwater Fish at the
Bioscience Center of Nagoya University.

Metric

14:

Acclimatization and Pretreatment
Conditions

Low

The study did not report whether test organisms were acclimatized and/or whether pre-
treatment conditions were the same for control and exposed groups.

Metric

15:

Number of Organisms and
Replicates per Group

Medium

20 test organisms with 3 replicates per treatment.

Continued on next page ...

Page 97 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 4292102 Table: 5 of 5

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Overall Duration: 11-21 days; Exposure Duration: 11-21 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Oryzias latipes\ d-rR strain; Embryo
Development/Growth
Tris(2-chloroethyl) phosphate (TCEP)

4292102

Domain

Metric

Rating

Comments

Domain 5: Outcome Assessment

Metric 16:

Metric 17:

Metric 18:

Adequacy of Test Conditions

Outcome Assessment Methodology

Consistency of Outcome
Assessment.

Low Housing and environmental conditions were adequate during incubation period. How-
ever, environmental conditions were not reported for test conditions.

High Assessment methods for body length, hatch % , incubation time, deformity % and heart

rate were reported.

High Outcome assessments were consistent across study groups.

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure

Low The study did not provide enough information to allow a comparison of environmental
conditions or other non-treatment-related factors across study groups.

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods High Appropriate statistics were used after data met assumptions of the tests.

Metric 22: Reporting of Data High Data for exposure-related findings were presented for each treatment and control group

and were adequate to determine values for hatchability, time to hatching, gross abnor-
mality rate, heart rate and body length.

Metric 23: Explanation of Unexpected Outcomes High No unexpected outcomes were reported.

Additional Comments: This evaluation form is relevant to development/growth outcomes (body length and percentage of gross abnormality rate) determined in medaka embryos
following exposure to TCEP.

Overall Quality Determination High

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 6310866 Table: 1 of 2

Study Citation:
Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Life Sciences Research Ltd, (1990). Fyrol CEF: Acute toxicity to rainbow trout.

Overall Duration: 0 - 4 days (0-96h); Exposure Duration: 0 - 4 days (0-96h)

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Salmo gairdnerr, Juvenile
Mortality

Tris(2-chloroethyl) phosphate (TCEP)

6310866

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1: Test Substance Identity
Metric 2: Test Substance Source
Metric 3: Test Substance Purity

High Name, CASNR, and structure provided

High Supplied by AKZO Chemical Inc., Batch no. 8101 K-l-4

Low Contrary to narrative, purity was not reported in appendix 2.

Domain 2: Test Design

Metric 4:

Negative Controls

High

Negative controls used.

Metric 5:

Negative Control Response

High

Zero mortality reported for controls

Metric 6:

Randomized Allocation

Medium

Random allocation was reported.

Domain 3: Exposure Characterization

Metric 7:

Experimental System/Test Media

Low

Only nominal concentrations used. Measures taken to account for volatility (e.g., head

Preparation

space, closed system etc.) not reported.

Metric 8:

Consistency of Exposure

High

exposures were consistent across groups.

Administration

Metric 9:

Measurement of Test Substance

Medium

Nominal concentrations were used.

Concentration

Metric 10:

Exposure Duration and Frequency

High

Exposure duration and frequency were appropriate for the test.

Metric 11:

Number of Exposure Groups/

High

Number of exposure groups and spacing appropriate for the test.

Spacing of Exposure Levels

Metric 12:

Testing at or Below Solubility Limit

High

Concentrations were below water solubility.

Domain 4: Test Organism

Metric 13:

Test Organism Characteristics

High

Fish and source were well described with the exception of sex.

Metric 14:

Acclimatization and Pretreatment

High

Acclimation was appropriate for test.

Conditions

Metric 15:

Number of Organisms and

Medium

Number of organisms and replicates were acceptable.

Replicates per Group

Domain 5: Outcome Assessment

Metric 16:

Adequacy of Test Conditions

High

Environmental conditions were adequate.

Metric 17:

Outcome Assessment Methodology

High

Intended outcomes were reported.

Metric 18:

Consistency of Outcome

High

Outcome assessments were consistent across groups.

Assessment.

Continued on next page ...

Page 99 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 6310866 Table: 1 of 2

Study Citation:
Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

... continued from previous page

Life Sciences Research Ltd, (1990). Fyrol CEF: Acute toxicity to rainbow trout.

Overall Duration: 0 - 4 days (0-96h); Exposure Duration: 0 - 4 days (0-96h)

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Salmo gairdnerr, Juvenile
Mortality

Tris(2-chloroethyl) phosphate (TCEP)

6310866

Domain

Metric

Rating

Comments

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure

High No confounding variables reported.
Medium No differences among groups reported.

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods

Metric 22: Reporting of Data

Metric 23: Explanation of Unexpected Outcomes

Low Statistical analyses were performed. However, statistics used were reported as not valid

for the data. Therefore approximate values were reported.

High Data reported for all outcomes.

High No unexpected outcomes reported.

Additional Comments: None

Overall Quality Determination

High

Page 100 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 6310866 Table: 2 of 2

Study Citation:
Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Life Sciences Research Ltd, (1990). Fyrol CEF: Acute toxicity to rainbow trout.

Overall Duration: 0 - 4 days (0-96h); Exposure Duration: 0 - 4 days (0-96h)

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Salmo gairdnerr, Juvenile
Mortality

Tris(2-chloroethyl) phosphate (TCEP)

6310866

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

Name, CASNR, and structure provided

Metric 2:

Test Substance Source

High

Supplied by AKZO Chemical Inc., Batch no. 8101 K-l-4

Metric 3:

Test Substance Purity

Low

Contrary to narrative, purity was not reported in appendix 2.

Domain 2: Test Design

Metric 4:

Negative Controls

High

Negative controls used.

Metric 5:

Negative Control Response

Low

No data on control conditions related to the outcome for this form (loss of coordination,

pigmentation, and edema) are reported or quantified.

Metric 6:

Randomized Allocation

Medium

Random allocation was reported.

Domain 3: Exposure Characterization

Metric 7:

Experimental System/Test Media

Low

Only nominal concentrations used. Measures taken to account for volatility (e.g., head

Preparation

space, closed system etc.) not reported.

Metric 8:

Consistency of Exposure

High

Exposures were consistent across groups.

Administration

Metric 9:

Measurement of Test Substance

Medium

Nominal concentrations were used.

Concentration

Metric 10:

Exposure Duration and Frequency

High

Exposure duration and frequency were appropriate for the test.

Metric 11:

Number of Exposure Groups/

High

Number of exposure groups and spacing appropriate for the test.

Spacing of Exposure Levels

Metric 12:

Testing at or Below Solubility Limit

High

Concentrations were below water solubility.

Domain 4: Test Organism

Metric 13:

Test Organism Characteristics

High

Fish and source were well described with the exception of sex.

Metric 14:

Acclimatization and Pretreatment

High

Acclimation was appropriate for test.

Conditions

Metric 15:

Number of Organisms and

Medium

Number of organisms and replicates were acceptable.

Replicates per Group

Domain 5: Outcome Assessment

Metric 16:

Adequacy of Test Conditions

High

Environmental conditions were adequate.

Metric 17:

Outcome Assessment Methodology

Low

This form is for observations reported in the results on loss of equilibrium, pigmenta-

tion, and edema (Page 18/24). This report has no data sheets to quantify these observa-

tions past report in the results. Responses for control individuals are not reported.

Metric 18:

Consistency of Outcome

Low

The methods do not report the frequency of when this outcome was recorded.

Assessment.

Continued on next page ...

Page 101 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 6310866 Table: 2 of 2

... continued from previous page

Study Citation:
Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Life Sciences Research Ltd, (1990). Fyrol CEF: Acute toxicity to rainbow trout.

Overall Duration: 0 - 4 days (0-96h); Exposure Duration: 0 - 4 days (0-96h)

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Vertebrate; Fish; Salmo gairdnerr, Juvenile
Mortality

Tris(2-chloroethyl) phosphate (TCEP)

6310866

Domain

Metric

Rating

Comments

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure

High	No confounding variables reported.

Medium No differences among groups reported.

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods	Low

Metric 22: Reporting of Data	Low

Metric 23: Explanation of Unexpected Outcomes	High

No Statistics for these observations were reported.

The data for the observations for this form are not presented.

No unexpected outcomes reported.

Additional Comments:

This form is for observations reported in the results on loss of equilibrium, pigmentation, and edema (Page 18/24). This report has no data sheets to quantify
these observations past report in the results. Responses for control individuals are not reported.	

Overall Quality Determination

Medium

Page 102 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469341 Table: 1 of 1

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Arukwe, A., Carteny, C. C., Eggen, T. (2016). Lipid peroxidation and oxidative stress responses in juvenile salmon exposed to waterborne levels of the
organophosphate compounds tris(2-butoxyethyl)- and tris(2-chloroethyl) phosphates. Journal of Toxicology and Environmental Health, Part A: Current
Issues 79(13-15):515-525.

Overall Duration: 4-10 days; Exposure Duration: 4-10 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)
Vertebrate; Fish; Salmo solar; Juvenile

Mechanistic-Cell signaling/function-Oxidative stress (including redox biology)

Tris(2-chloroethyl) phosphate (TCEP)

5469341

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1: Test Substance Identity
Metric 2: Test Substance Source

Metric 3: Test Substance Purity

High Th tests substance was identified by chemical name and molecular formula.

Low The test substance was obtained from obtained from Sigma-Aldrich Chemie GmbH but

its was not analytically verified by the performing laboratory.

High Percent purity of TCEP was reported as 97% .

Domain 2: Test Design

Metric 4: Negative Controls
Metric 5: Negative Control Response
Metric 6: Randomized Allocation

High Study authors reported using an appropriate concurrent negative control group.
High The biological responses of the negative control group were adequate.
Low Researchers did not report how organisms were allocated to study groups.

Domain 3: Exposure Characterization

Metric 7:

Experimental System/Test Media

Medium

The experimental system and test media preparation methods were reported. A

Preparation

semistatic experimental protocol was used. Test solution were prepared in Milli-Q water.

Test solutions were renewed every 3 days.

Metric 8:

Consistency of Exposure

High

Details of exposure administration were reported and exposures were administered

Administration

consistently across study groups.

Metric 9:

Measurement of Test Substance

Low

Exposure concentrations were not measured.

Concentration

Metric 10:

Exposure Duration and Frequency

High

7-d exposure duration is appropriate for the study type.

Metric 11:

Number of Exposure Groups/

High

Three exposure groups (0.04, 0.2, or 1 mg/L) and a control were used and adequate to

Spacing of Exposure Levels

address the purpose of the study.

Metric 12:

Testing at or Below Solubility Limit

High

Exposure concentrations were below the water solubility limit.

Domain 4: Test Organism

Metric 13: Test Organism Characteristics

Metric 14: Acclimatization and Pretreatment

Conditions
Metric 15: Number of Organisms and
Replicates per Group

High The test organisms were adequately described. Average length and weight measure-
ments were provided. Test organisms were obtained from Settefiskanlegget Lundamo
AS (Lundamo, Norway).

High The test organisms were acclimatized to test conditions for 9 days and all pretreatment

conditions were the same for control and exposed organisms
Low The numbers of test organisms was reported. "Fish were divided into 6 treatments and
1 control where each group constitutes 16 individuals." The use of replicates was not
reported.

Continued on next page ...

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469341 Table: 1 of 1

... continued from previous page

Overall Duration: 4-10 days; Exposure Duration: 4-10 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Vertebrate; Fish; Salmo solar; Juvenile

Mechanistic-Cell signaling/function-Oxidative stress (including redox biology)

Tris(2-chloroethyl) phosphate (TCEP)

5469341

Domain

Metric

Rating

Comments

Domain 5: Outcome Assessment

Metric 16: Adequacy of Test Conditions

Metric 17: Outcome Assessment Methodology

Metric 18: Consistency of Outcome
Assessment

High Organism housing and environmental conditions at which the tanks were maintained
were appropriate. "The tanks were kept at a constant temperature of 8°C and a 12:12-h
photoperiod." However, water quality parameters were (pH, DO, etc.) were not moni-
tored.

High The outcome assessment methodology addressed or reported the intended outcome(s)
of interest. Methodologies to determine gene expression of GPx, GR, GST, peroxisome
proliferator-activated receptors (PPAR) were described well. The immunochemical
method to determine presence of PPAR proteins was also described well.

High Details of the outcome assessment protocol were reported, and outcomes were assessed
consistently across study groups.

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test Low The study did not provide enough information to allow a comparison of environmental

Design and Procedures conditions or other non-treatment-related factors across study groups.

Metric 20: Outcomes Unrelated to Exposure Medium There was no information in the study to suggest differences among groups in animal

attrition or health outcomes unrelated to exposure (e.g., infection) that could influence
the outcome assessment.

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods High Statistical methods were adequately described. "One-way analysis of variance

(ANOVA) was performed, followed by post hoc analysis (Duncan"s test) between sub-
groups."

Metric 22: Reporting of Data Medium Data for exposure-related findings were presented for each treatment and control group

for gene expression data. The immunoblot analysis results (Figure ID) is missing in the
manuscript.

Metric 23: Explanation of Unexpected Outcomes High There were no unexpected outcomes

Additional Comments: This form includes all mechanistic data- gene expression of glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST),
peroxisome proliferator-activated receptors (PPAR), and presence of PPAR proteins using immunochemical methods.

Overall Quality Determination High

Page 104 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5184752 Table: 1 of 1

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Kovacevic, V., Simpson, A. J., Simpson, M. J. (2018). Investigation of daphnia magna sub-lethal exposure to organophosphate esters in the presence of
dissolved organic matter using "H NMR-based metabolomics. Metabolites 8(2):34.

Overall Duration: 0 - 4 days (0-96h); Exposure Duration: 0 - 4 days (0-96h)

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)
Invertebrate; Arthropods; Daphnia magna; Adult

Mechanistic-Biomarkers (exposure and effect)-Cell signaling/function-Nutritional and Metabolic

Tris(2-chloroethyl) phosphate (TCEP)

5184752

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

Chemical was identified by name and chemical structure (Table 1).

Metric 2:

Test Substance Source

Low

The test substance identity was not analytically verified by the performing laboratory.

Metric 3:

Test Substance Purity

High

Chemical purity reported as 97% .

Domain 2: Test Design

Metric 4:

Negative Controls

High

Study authors reported using an appropriate concurrent negative control group, both

DOM and clean controls were used.

Metric 5:

Negative Control Response

Medium

The biological response of the negative control groups was reported for analyzed

metabolites but not for survival.

Metric 6:

Randomized Allocation

Low

Researchers did not report how organisms were allocated to study groups.

Domain 3: Exposure Characterization

Metric 7:

Experimental System/Test Media

High

The experimental system and methods for preparation of test media were described in

Preparation

adequate detail. Stock solution was prepared in dechlorinated water and beakers with

test solution "were sealed with parafilm and equilibrated on a magnetic stirrer at room

temperature in the dark for 48 h."

Metric 8:

Consistency of Exposure

High

Exposures were administered consistently across study groups.

Administration

Metric 9:

Measurement of Test Substance

High

Exposure concentrations were measured using appropriate analytical technologies and

Concentration

methods, and were similar to nominals.

Metric 10:

Exposure Duration and Frequency

High

The duration of exposure was reported (48 hours) and appropriate for the study type.

Metric 11:

Number of Exposure Groups/

N/A

Only one concentration was tested.

Spacing of Exposure Levels

Metric 12:

Testing at or Below Solubility Limit

High

Exposure concentration was below the water solubility limit.

Domain 4: Test Organism

Metric 13:

Test Organism Characteristics

High

The test organisms were adequately described and were obtained from a reliable source.

Laboratory cultured adult daphnids (16 days old) were used.

Metric 14:

Acclimatization and Pretreatment

High

All pretreatment conditions were the same for control and exposed organisms.

Conditions

Metric 15:

Number of Organisms and

Medium

The numbers of test organisms and replicates were reported, 10 replicates with 10 daph-

Replicates per Group

nids each.

Continued on next page ...

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5184752 Table: 1 of 1

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Overall Duration: 0 - 4 days (0-96h); Exposure Duration: 0 - 4 days (0-96h)

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)
Invertebrate; Arthropods; Daphnia magna; Adult

Mechanistic-Biomarkers (exposure and effect)-Cell signaling/function-Nutritional and Metabolic

Tris(2-chloroethyl) phosphate (TCEP)

5184752

Domain

Metric

Rating

Comments

Domain 5: Outcome Assessment

Metric 16: Adequacy of Test Conditions

Metric 17: Outcome Assessment Methodology

Metric 18: Consistency of Outcome
Assessment.

Medium Daphnia culture conditions were reported. Organism housing and feeding during expo-
sure period were reported. Biomass loading (1 daphnid/30 ml) was appropriate. It was
stated that temperature and light conditions were the same as the culture conditions but
specifics during the exposure period were not reported.

High The outcome assessment methodology reported the intended outcome of interest.
Metabolomics methods were described well.

High Outcomes were assessed consistently across study groups.

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test Medium

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure Medium

There were no reported differences among the study groups in environmental conditions
although specifics were not reported.

There was no information in the study to suggest differences among groups in animal
attrition or health outcomes unrelated to exposure (e.g., infection) that could influence
the outcome assessment.

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods High Statistical methods were adequately described. "The statistical significance of the in-

dividual metabolite percent changes was determined with a t-test (two-tailed, equal
variances, p < 0.05) and the results of this statistical analysis is given in Table S3 in the
Supplementary Materials."

Metric 22: Reporting of Data High Table S3 has statistical results for all treatments.

Metric 23: Explanation of Unexpected Outcomes High Unexpected outcomes were satisfactorily explained

Additional Comments: The study examined the effect of dissolved organic matter (DOM) on the sub-lethal toxicity of TCEP to D. magna using 1H NMR-based metabolomics.
TCEP was tested alone and in the presence of DOM.

Overall Quality Determination High

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 10064285 Table: 1 of 6

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Zhang, S., Hagstrom, D., Hayes, P., Graham, A., Collins, E. S. (2019). Multi-behavioral endpoint testing of an 87-chemical compound library in freshwater

planarians. Toxicological Sciences 167(1 ):26-44.

Overall Duration: 4-10 days; Exposure Duration: 4-10 days

Aquatic (freshwater); Cell Culture Media; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact
uptake route)

Invertebrate; Worms (e.g., Annelids, Nematodes); Dugesia japonica\ Adult
Mortality

Tris(2-chloroethyl) phosphate (TCEP)

10064285

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

Identified by name and CASRN in supplemental notes.

Metric 2:

Test Substance Source

Low

Source of stock solutions were identified. Analytical verification not reported.

Metric 3:

Test Substance Purity

Low

Purity or grade was not reported.

Domain 2: Test Design

Metric 4:

Negative Controls

High

Solvent controls were reported.

Metric 5:

Negative Control Response

High

Negative control response acceptable and presented within the supplemental excel file.

Metric 6:

Randomized Allocation

Low

Worms were selected by specified size.

Domain 3: Exposure Characterization

Metric 7:

Experimental System/Test Media

High

Experimental system well described.

Preparation

Metric 8:

Consistency of Exposure

High

Exposures were consistent across groups.

Administration

Metric 9:

Measurement of Test Substance

Low

Stock solutions of different concentrations were purchased. No analytical measurements

Concentration

were reported.

Metric 10:

Exposure Duration and Frequency

High

Exposure concentration and frequency were appropriate for the test.

Metric 11:

Number of Exposure Groups/

High

Number of exposure groups and spacing were appropriate for the tests.

Spacing of Exposure Levels

Metric 12:

Testing at or Below Solubility Limit

High

The highest concentration (100 uM) is 28 mg/L which is over the solubility listed in the

final scope (7.9 mg/L).

Domain 4: Test Organism

Metric 13:

Test Organism Characteristics

High

Test organisms were well described.

Metric 14:

Acclimatization and Pretreatment

High

Acclimation and pretreatments were appropriate for tests.

Conditions

Metric 15:

Number of Organisms and

Medium

Number of organisms and replicates were acceptable.

Replicates per Group

Domain 5: Outcome Assessment

Metric 16:

Adequacy of Test Conditions

High

Test conditions were adequate.

Metric 17:

Outcome Assessment Methodology

High

Intended outcomes reported in supplemental excel file.

Continued on next page ...

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 10064285 Table: 1 of 6

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Zhang, S., Hagstrom, D., Hayes, P., Graham, A., Collins, E. S. (2019). Multi-behavioral endpoint testing of an 87-chemical compound library in freshwater

planarians. Toxicological Sciences 167(1 ):26-44.

Overall Duration: 4-10 days; Exposure Duration: 4-10 days

Aquatic (freshwater); Cell Culture Media; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact
uptake route)

Invertebrate; Worms (e.g., Annelids, Nematodes); Dugesia japonica\ Adult
Mortality

Tris(2-chloroethyl) phosphate (TCEP)

10064285

Domain

Metric

Rating Comments

Metric 18:

Consistency of Outcome
Assessment

High Outcome methodology was consistent.

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure

High No confounding variables reported.
Medium No unrelated outcomes were reported.

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods

Metric 22: Reporting of Data

Metric 23: Explanation of Unexpected Outcomes

High Statistics well described.

High Data reported in supplemental excel file.

High There were no unexpected outcomes reported.

Additional Comments: supplementary data (table 2) provides LC50 values for 12 day worms but TCEP is not listed in this data. Specific data on mortality for TCEP is within the
excel file (no mortality for worms at concentrations in this work).

Overall Quality Determination

High

Page 108 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 10064285 Table: 2 of 6

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Zhang, S., Hagstrom, D., Hayes, P., Graham, A., Collins, E. S. (2019). Multi-behavioral endpoint testing of an 87-chemical compound library in freshwater

planarians. Toxicological Sciences 167(1 ):26-44.

Overall Duration: 4-10 days; Exposure Duration: 4-10 days

Aquatic (freshwater); Cell Culture Media; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact
uptake route)

Invertebrate; Worms (e.g., Annelids, Nematodes); Dugesia japonica\ Adult
Development/Growth
Tris(2-chloroethyl) phosphate (TCEP)

10064285

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

Identified by name and CASRN

Metric 2:

Test Substance Source

Low

Source of stock solutions were identified. Analytical verification not reported.

Metric 3:

Test Substance Purity

Low

Purity or grade was not reported.

Domain 2: Test Design

Metric 4:

Negative Controls

High

Solvent controls were reported.

Metric 5:

Negative Control Response

High

Negative control response acceptable.

Metric 6:

Randomized Allocation

Low

Worms were selected by specified size.

Domain 3: Exposure Characterization

Metric 7:

Experimental System/Test Media

High

Experimental system well described.

Preparation

Metric 8:

Consistency of Exposure

High

Exposures were consistent across groups.

Administration

Metric 9:

Measurement of Test Substance

Low

Stock solutions of different concentrations were purchased. No analytical measurements

Concentration

were reported.

Metric 10:

Exposure Duration and Frequency

High

Exposure concentration and frequency were appropriate for the test.

Metric 11:

Number of Exposure Groups/

High

Number of exposure groups and spacing were appropriate for the tests.

Spacing of Exposure Levels

Metric 12:

Testing at or Below Solubility Limit

Medium

The highest concentration (100 uM) is 28 mg/L which is over the solubility listed in the

final scope (7.9 mg/L).

Domain 4: Test Organism

Metric 13:

Test Organism Characteristics

High

Test organisms were well described.

Metric 14:

Acclimatization and Pretreatment

High

Acclimation and pretreatments were appropriate for tests.

Conditions

Metric 15:

Number of Organisms and

Medium

Number of organisms and replicates were acceptable.

Replicates per Group

Domain 5: Outcome Assessment

Metric 16:

Adequacy of Test Conditions

High

Test conditions were adequate.

Metric 17:

Outcome Assessment Methodology

High

Intended outcomes were reported.

Metric 18:

Consistency of Outcome

High

Outcome methodology was consistent.

Assessment.

Continued on next page ...

Page 109 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 10064285 Table: 2 of 6

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Zhang, S., Hagstrom, D., Hayes, P., Graham, A., Collins, E. S. (2019). Multi-behavioral endpoint testing of an 87-chemical compound library in freshwater

planarians. Toxicological Sciences 167(1 ):26-44.

Overall Duration: 4-10 days; Exposure Duration: 4-10 days

Aquatic (freshwater); Cell Culture Media; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact
uptake route)

Invertebrate; Worms (e.g., Annelids, Nematodes); Dugesia japonica\ Adult
Development/Growth
Tris(2-chloroethyl) phosphate (TCEP)

10064285

Domain Metric

Rating

Comments

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure

High
Medium

No confounding variables reported.
No unrelated outcomes were reported.

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods

Metric 22: Reporting of Data

Metric 23: Explanation of Unexpected Outcomes

High
Low
High

Statistics well described.

Data were reported in supplemental, however reporting of data was not clear.
There were no unexpected outcomes reported.

Additional Comments: This form represents measures of growth, specifically eye regeneration on day 7. Data is located in the supplementary excel file.

Overall Quality Determination

High

Page 110 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 10064285 Table: 3 of 6

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Zhang, S., Hagstrom, D., Hayes, P., Graham, A., Collins, E. S. (2019). Multi-behavioral endpoint testing of an 87-chemical compound library in freshwater

planarians. Toxicological Sciences 167(1 ):26-44.

Overall Duration: 4-10 days; Exposure Duration: 4-10 days

Aquatic (freshwater); Cell Culture Media; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact
uptake route)

Invertebrate; Worms (e.g., Annelids, Nematodes); Dugesia japonica\ Adult
Behavioral

Tris(2-chloroethyl) phosphate (TCEP)

10064285

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

Identified by name and CASRN

Metric 2:

Test Substance Source

Low

Source of stock solutions were identified. Analytical verification not reported.

Metric 3:

Test Substance Purity

Low

Purity or grade was not reported.

Domain 2: Test Design

Metric 4:

Negative Controls

High

Solvent controls were reported.

Metric 5:

Negative Control Response

High

Negative control response acceptable.

Metric 6:

Randomized Allocation

Low

Worms were selected by specified size.

Domain 3: Exposure Characterization

Metric 7:

Experimental System/Test Media

High

Experimental system well described.

Preparation

Metric 8:

Consistency of Exposure

High

Exposures were consistent across groups.

Administration

Metric 9:

Measurement of Test Substance

Low

Stock solutions of different concentrations were purchased. No analytical measurements

Concentration

were reported.

Metric 10:

Exposure Duration and Frequency

High

Exposure concentration and frequency were appropriate for the test.

Metric 11:

Number of Exposure Groups/

High

Number of exposure groups and spacing were appropriate for the tests.

Spacing of Exposure Levels

Metric 12:

Testing at or Below Solubility Limit

Medium

The highest concentration (100 uM) is 28 mg/L which is over the solubility listed in the

final scope (7.9 mg/L).

Domain 4: Test Organism

Metric 13:

Test Organism Characteristics

High

Test organisms were well described.

Metric 14:

Acclimatization and Pretreatment

High

Acclimation and pretreatments were appropriate for tests.

Conditions

Metric 15:

Number of Organisms and

Medium

Number of organisms and replicates were acceptable.

Replicates per Group

Domain 5: Outcome Assessment

Metric 16:

Adequacy of Test Conditions

High

Test conditions were adequate.

Metric 17:

Outcome Assessment Methodology

High

Intended outcomes were reported.

Metric 18:

Consistency of Outcome

High

Outcome methodology was consistent.

Assessment.

Continued on next page ...

Page 111 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 10064285 Table: 3 of 6

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Zhang, S., Hagstrom, D., Hayes, P., Graham, A., Collins, E. S. (2019). Multi-behavioral endpoint testing of an 87-chemical compound library in freshwater

planarians. Toxicological Sciences 167(1 ):26-44.

Overall Duration: 4-10 days; Exposure Duration: 4-10 days

Aquatic (freshwater); Cell Culture Media; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact
uptake route)

Invertebrate; Worms (e.g., Annelids, Nematodes); Dugesia japonica\ Adult
Behavioral

Tris(2-chloroethyl) phosphate (TCEP)

10064285

Domain Metric

Rating

Comments

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure

High
Medium

No confounding variables reported.
No unrelated outcomes were reported.

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods

Metric 22: Reporting of Data

Metric 23: Explanation of Unexpected Outcomes

High
High
High

Statistics well described.

Data were reported in supplemental excel file.

There were no unexpected outcomes reported.

Additional Comments: This form is for the behavior metrics which on day 7
mentary Excel file.

are unstimulated behavior and phototaxsis. The TCEP data for this metric is located in the supple-

Overall Quality Determination

High

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 10064285 Table: 4 of 6

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Zhang, S., Hagstrom, D., Hayes, P., Graham, A., Collins, E. S. (2019). Multi-behavioral endpoint testing of an 87-chemical compound library in freshwater

planarians. Toxicological Sciences 167(1 ):26-44.

Overall Duration: 11-21 days; Exposure Duration: 11-21 days

Aquatic (freshwater); Cell Culture Media; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact
uptake route)

Invertebrate; Worms (e.g., Annelids, Nematodes); Dugesia japonica\ Adult
Mortality

Tris(2-chloroethyl) phosphate (TCEP)

10064285

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

Identified by name and CASRN

Metric 2:

Test Substance Source

Low

Source of stock solutions were identified. Analytical verification not reported.

Metric 3:

Test Substance Purity

Low

Purity or grade was not reported.

Domain 2: Test Design

Metric 4:

Negative Controls

High

Solvent controls were reported.

Metric 5:

Negative Control Response

High

Negative control response acceptable.

Metric 6:

Randomized Allocation

Low

Worms were selected by specified size.

Domain 3: Exposure Characterization

Metric 7:

Experimental System/Test Media

High

Experimental system well described.

Preparation

Metric 8:

Consistency of Exposure

High

Exposures were consistent across groups.

Administration

Metric 9:

Measurement of Test Substance

Low

Stock solutions of different concentrations were purchased. No analytical measurements

Concentration

were reported.

Metric 10:

Exposure Duration and Frequency

High

Exposure concentration and frequency were appropriate for the test.

Metric 11:

Number of Exposure Groups/

High

Number of exposure groups and spacing were appropriate for the tests.

Spacing of Exposure Levels

Metric 12:

Testing at or Below Solubility Limit

Medium

The highest concentration (100 uM) is 28 mg/L which is over the solubility listed in the

final scope (7.9 mg/L).

Domain 4: Test Organism

Metric 13:

Test Organism Characteristics

High

Test organisms were well described.

Metric 14:

Acclimatization and Pretreatment

High

Acclimation and pretreatments were appropriate for tests.

Conditions

Metric 15:

Number of Organisms and

Medium

Number of organisms and replicates were acceptable.

Replicates per Group

Domain 5: Outcome Assessment

Metric 16:

Adequacy of Test Conditions

High

Test conditions were adequate.

Metric 17:

Outcome Assessment Methodology

High

Intended outcomes reported in supplemental excel file.

Metric 18:

Consistency of Outcome

High

Outcome methodology was consistent.

Assessment.

Continued on next page ...

Page 113 of 170

-------
Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 10064285 Table: 4 of 6

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Zhang, S., Hagstrom, D., Hayes, P., Graham, A., Collins, E. S. (2019). Multi-behavioral endpoint testing of an 87-chemical compound library in freshwater

planarians. Toxicological Sciences 167(1 ):26-44.

Overall Duration: 11-21 days; Exposure Duration: 11-21 days

Aquatic (freshwater); Cell Culture Media; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact
uptake route)

Invertebrate; Worms (e.g., Annelids, Nematodes); Dugesia japonica\ Adult
Mortality

Tris(2-chloroethyl) phosphate (TCEP)

10064285

Domain Metric

Rating

Comments

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure

High
Medium

No confounding variables reported.
No unrelated outcomes were reported.

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods

Metric 22: Reporting of Data

Metric 23: Explanation of Unexpected Outcomes

High
High
High

Statistics well described.

Data reported in supplemental excel file.

There were no unexpected outcomes reported.

Additional Comments: supplementary data (table 2) provides LC50 values for 12 day worms but TCEP is not listed in this data. Specific data on mortality for TCEP is within the
excel file (no mortality for worms at concentrations in this work).

Overall Quality Determination

High



Page 114 of 170

-------
Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 10064285 Table: 5 of 6

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Zhang, S., Hagstrom, D., Hayes, P., Graham, A., Collins, E. S. (2019). Multi-behavioral endpoint testing of an 87-chemical compound library in freshwater

planarians. Toxicological Sciences 167(1 ):26-44.

Overall Duration: 11-21 days; Exposure Duration: 11-21 days

Aquatic (freshwater); Cell Culture Media; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact
uptake route)

Invertebrate; Worms (e.g., Annelids, Nematodes); Dugesia japonica\ Adult
Development/Growth
Tris(2-chloroethyl) phosphate (TCEP)

10064285

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

Identified by name and CASRN

Metric 2:

Test Substance Source

Low

Source of stock solutions were identified. Analytical verification not reported.

Metric 3:

Test Substance Purity

Low

Purity or grade was not reported.

Domain 2: Test Design

Metric 4:

Negative Controls

High

Solvent controls were reported.

Metric 5:

Negative Control Response

High

Negative control response acceptable.

Metric 6:

Randomized Allocation

Low

Worms were selected by specified size.

Domain 3: Exposure Characterization

Metric 7:

Experimental System/Test Media

High

Experimental system well described.

Preparation

Metric 8:

Consistency of Exposure

High

Exposures were consistent across groups.

Administration

Metric 9:

Measurement of Test Substance

Low

Stock solutions of different concentrations were purchased. No analytical measurements

Concentration

were reported.

Metric 10:

Exposure Duration and Frequency

High

Exposure concentration and frequency were appropriate for the test.

Metric 11:

Number of Exposure Groups/

High

Number of exposure groups and spacing were appropriate for the tests.

Spacing of Exposure Levels

Metric 12:

Testing at or Below Solubility Limit

Medium

The highest concentration (100 uM) is 28 mg/L which is over the solubility listed in the

final scope (7.9 mg/L).

Domain 4: Test Organism

Metric 13:

Test Organism Characteristics

High

Test organisms were well described.

Metric 14:

Acclimatization and Pretreatment

High

Acclimation and pretreatments were appropriate for tests.

Conditions

Metric 15:

Number of Organisms and

Medium

Number of organisms and replicates were acceptable.

Replicates per Group

Domain 5: Outcome Assessment

Metric 16:

Adequacy of Test Conditions

High

Test conditions were adequate.

Metric 17:

Outcome Assessment Methodology

High

Intended outcomes were reported.

Metric 18:

Consistency of Outcome

High

Outcome methodology was consistent.

Assessment.

Continued on next page ...

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December 2023

Environmental Hazard Evaluation

HERO ID: 10064285 Table: 5 of 6

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Zhang, S., Hagstrom, D., Hayes, P., Graham, A., Collins, E. S. (2019). Multi-behavioral endpoint testing of an 87-chemical compound library in freshwater

planarians. Toxicological Sciences 167(1 ):26-44.

Overall Duration: 11-21 days; Exposure Duration: 11-21 days

Aquatic (freshwater); Cell Culture Media; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact
uptake route)

Invertebrate; Worms (e.g., Annelids, Nematodes); Dugesia japonica\ Adult
Development/Growth
Tris(2-chloroethyl) phosphate (TCEP)

10064285

Domain Metric

Rating

Comments

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure

High
Medium

No confounding variables reported.
No unrelated outcomes were reported.

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods

Metric 22: Reporting of Data

Metric 23: Explanation of Unexpected Outcomes

High
Low
High

Statistics well described.

Data were reported in supplemental, however reporting of data was not clear.
There were no unexpected outcomes reported.

Additional Comments: This form identifies morphology assessments at day 12 of exposure. This is identified in Figure one of the paper. Results are located on page 9/18, TCEP
was not. detailed for these data.

Overall Quality Determination

High

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December 2023

Environmental Hazard Evaluation

HERO ID: 10064285 Table: 6 of 6

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Zhang, S., Hagstrom, D., Hayes, P., Graham, A., Collins, E. S. (2019). Multi-behavioral endpoint testing of an 87-chemical compound library in freshwater

planarians. Toxicological Sciences 167(1 ):26-44.

Overall Duration: 11-21 days; Exposure Duration: 11-21 days

Aquatic (freshwater); Cell Culture Media; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact
uptake route)

Invertebrate; Worms (e.g., Annelids, Nematodes); Dugesia japonica\ Adult
Behavioral

Tris(2-chloroethyl) phosphate (TCEP)

10064285

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

Identified by name and CASRN

Metric 2:

Test Substance Source

Low

Source of stock solutions were identified. Analytical verification not reported.

Metric 3:

Test Substance Purity

Low

Purity or grade was not reported.

Domain 2: Test Design

Metric 4:

Negative Controls

High

Solvent controls were reported.

Metric 5:

Negative Control Response

High

Negative control response acceptable.

Metric 6:

Randomized Allocation

Low

Worms were selected by specified size.

Domain 3: Exposure Characterization

Metric 7:

Experimental System/Test Media

High

Experimental system well described.

Preparation

Metric 8:

Consistency of Exposure

High

Exposures were consistent across groups.

Administration

Metric 9:

Measurement of Test Substance

Low

Stock solutions of different concentrations were purchased. No analytical measurements

Concentration

were reported.

Metric 10:

Exposure Duration and Frequency

High

Exposure concentration and frequency were appropriate for the test.

Metric 11:

Number of Exposure Groups/

High

Number of exposure groups and spacing were appropriate for the tests.

Spacing of Exposure Levels

Metric 12:

Testing at or Below Solubility Limit

Medium

The highest concentration (100 uM) is 28 mg/L which is over the solubility listed in the

final scope (7.9 mg/L).

Domain 4: Test Organism

Metric 13:

Test Organism Characteristics

High

Test organisms were well described.

Metric 14:

Acclimatization and Pretreatment

High

Acclimation and pretreatments were appropriate for tests.

Conditions

Metric 15:

Number of Organisms and

Medium

Number of organisms and replicates were acceptable.

Replicates per Group

Domain 5: Outcome Assessment

Metric 16:

Adequacy of Test Conditions

High

Test conditions were adequate.

Metric 17:

Outcome Assessment Methodology

High

Intended outcomes were reported.

Metric 18:

Consistency of Outcome

High

Outcome methodology was consistent.

Assessment.

Continued on next page ...

Page 117 of 170

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December 2023

Environmental Hazard Evaluation

HERO ID: 10064285 Table: 6 of 6

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Zhang, S., Hagstrom, D., Hayes, P., Graham, A., Collins, E. S. (2019). Multi-behavioral endpoint testing of an 87-chemical compound library in freshwater

planarians. Toxicological Sciences 167(1 ):26-44.

Overall Duration: 11-21 days; Exposure Duration: 11-21 days

Aquatic (freshwater); Cell Culture Media; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact
uptake route)

Invertebrate; Worms (e.g., Annelids, Nematodes); Dugesia japonica\ Adult
Behavioral

Tris(2-chloroethyl) phosphate (TCEP)

10064285

Domain Metric

Rating

Comments

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure

High
Medium

No confounding variables reported.
No unrelated outcomes were reported.

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods

Metric 22: Reporting of Data

Metric 23: Explanation of Unexpected Outcomes

High
High
High

Statistics well described.

Data were reported in supplemental excel file.

There were no unexpected outcomes reported.

Additional Comments: This form represents behavior metrics in the paper that include: unstimulated behavior, thermotaxsis, phototaxis, and scrunching. These data for TCEP are
located in the supplementary excel file.

Overall Quality Determination

High

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469417 Table: 1 of 6

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Zhang, S., Ireland, D., Sipes, N. S., Behl, M., Collins, E. S. (2019). Screening for neurotoxic potential of 15 flame retardants using freshwater planarians.

Neurotoxicology and Teratology 7354-66.

Overall Duration: 11-21 days; Exposure Duration: 11-21 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Invertebrate; Worms (e.g., Annelids, Nematodes); Dugesia japonica\ regenerative; Adult
Mortality

Tris(2-chloroethyl) phosphate (TCEP)

5469417

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1: Test Substance Identity
Metric 2: Test Substance Source

Metric 3: Test Substance Purity

High clearly identified with name and CAS #

Low Sourced from Sigma Aldrich, but chemical was not analytically verified by the labora-

tory.

High reported as 99.07% pure

Domain 2: Test Design

Metric 4: Negative Controls

Metric 5: Negative Control Response
Metric 6: Randomized Allocation

Low Authors did not use a true negative control. "Negative" controls contained either ac-

etaminophen or L-ascorbic acid. A positive solvent control (0.5% DMSO) was also
used. The authors presented information from previous studies to verify that ac-
etaminophen or L-ascorbic acid did not affect the test organisms.

High 100% survival in solvent control (0.5% DMSO), no mortality observed in "negative"

controls

Low No mention of randomization in this paper. Author did cite another paper which may

have described randomization.

Domain 3: Exposure Characterization
Metric 7:

Metric 8:
Metric 9:
Metric 10:
Metric 11:

Experimental System/Test Media
Preparation

Consistency of Exposure
Administration

Measurement of Test Substance
Concentration

Exposure Duration and Frequency

Number of Exposure Groups/
Spacing of Exposure Levels

Metric 12: Testing at or Below Solubility Limit

Low The experimental system and methods for preparation of test media were described in

adequate detail and appropriately accounted for the physical-chemical properties of the
test substance (solvent used due to the low solubility of the test material). Measured test
concentrations were not provided.

High No inconsistencies were noticed or reported

Low Exposure concentrations were not measured, highest concentration listed as 99.5 uM in

supplemental data instead of a nominal value of 100 uM as stated in text

High Exposure duration (12 days) was adequate to cause mortality for some of the chemicals

in this study (but not TCEP).

High Exposure concentrations seemed adequate to illicit a response (mortality) for most

chemicals tested in this study. This was below the NOAEL for TCEP, but the purpose
of the study was to compare the mortality between chemicals within a specific group, so
this was not determined to affect the rating of this study.

High Solvent (0.5% DMSO) was used to facilitate the dissolution of the test material into the

test medium.

Domain 4: Test Organism

Continued on next page .

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469417 Table: 1 of 6

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Zhang, S., Ireland, D., Sipes, N. S., Behl, M., Collins, E. S. (2019). Screening for neurotoxic potential of 15 flame retardants using freshwater planarians.

Neurotoxicology and Teratology 7354-66.

Overall Duration: 11-21 days; Exposure Duration: 11-21 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Invertebrate; Worms (e.g., Annelids, Nematodes); Dugesia japonica\ regenerative; Adult
Mortality

Tris(2-chloroethyl) phosphate (TCEP)

5469417

Domain	Metric	Rating	Comments

Metric 13:

Test Organism Characteristics

High

Supplemental methods description describes the test organism in more detail (Zhang
et al., 2019). Test organisms were described as adults and "Test worms were manually
selected to fall within a certain range of sizes and we found full worm length, after au-
tomated size measurement, to be 7.3 6 2.3 mm (mean 6 SD), and tail worm length to be
7.3 mm 2.7 mm (mean 6 SD).

Metric 14:

Acclimatization and Pretreatment
Conditions

High

In supplemental materials, pretreatment procedure was described as: "Animals were
starved for at least 5 days before beingused for experiments and their containers were
cleaned immediately prior to worm selection for experiments." This was adequate for
gut-clearance.

Metric 15:

Number of Organisms and
Replicates per Group

Medium

8 individuals were used for each replicate.

Domain 5: Outcome Assessment







Metric 16:

Adequacy of Test Conditions

Low

Test conditions were fully described in supplemental methods paper (Zhang et al.,
2019): "Wells of a 48- well plate with 200 ml of IO water." The authors noted that in
the present study, they updated the feeding procedure, but this was not described clearly,
"we fed planarians used in FR screen 2 commercial freeze-dried organic chicken liver
(Amazon, Seattle, WA) to better control food quality and thus minimize animal fitness
variability." It was unclear when this supplemental feeding occurred during the exposure
period or if the lack of feeding could have contributed to additional mortality in some
chemicals in the screen (No mortality was observed for TCEP, so this was not an issue
for this chemcial).

Metric 17:

Outcome Assessment Methodology

High

Outcome assessment was adequate to describe the comparative mortality of several
flame retardants following exposure to the test material.

Metric 18:

Consistency of Outcome

High

assessment methods seemed consistent across groups

Assessment.

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test
Design and Procedures

Metric 20: Outcomes Unrelated to Exposure

Low	The study did not provide enough information to allow a comparison of environmental

conditions or other non-treatment-related factors across study groups. Besides the feed-
ing procedure, which did not contribute to mortality for TCEP, there were no reported
differences among the study groups in environmental conditions or other factors that
could influence the outcome assessment.

Medium There was no information in the study to suggest differences among groups in animal
attrition or health outcomes unrelated to exposure (e.g., infection) that could influence
the outcome assessment.

Domain 7: Data Presentation and Analysis

Continued on next page ...

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469417 Table: 1 of 6

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Zhang, S., Ireland, D., Sipes, N. S., Behl, M., Collins, E. S. (2019). Screening for neurotoxic potential of 15 flame retardants using freshwater planarians.

Neurotoxicology and Teratology 7354-66.

Overall Duration: 11-21 days; Exposure Duration: 11-21 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Invertebrate; Worms (e.g., Annelids, Nematodes); Dugesia japonica\ regenerative; Adult
Mortality

Tris(2-chloroethyl) phosphate (TCEP)

5469417

Domain

Metric

Rating

Comments

Metric 21:

Statistical Methods

Low

The authors describe the statistical procedure as: "Statistical significance was deter-
mined using either a one-tailed Fisher's exact test for lethality, eye regeneration, photo-
taxis, and scrunching endpoints; Mann Whitney U test for thermotaxis and unstimulated
behavior; or two-tailed t-test for unstimulated behavior (depending on normality of the
sample) using a significance level of 0.05. This was adequate to determine if mortality
was significant." This analysis and the raw data describing mortality were not presented
in this study.

Metric 22:

Reporting of Data

Low

A large amount of data was presented in the supplemental file (Zhang et al., 2019):
https://doi.Org/10.1016/j.ntt.2019.03.003. Data were not clear and interpretation was
difficult. It appeared that no mortality was observed in the adults following exposure to
TCEP for 7-12 days, but this was not clearly discussed.

Metric 23:

Explanation of Unexpected Outcomes

High

no mortalities for entire test duration in any treatment. The lack of bioactivity observed
in this study for TCEP was not consistent with previous studies showing bioactivity in
Zebrafish. This was explained in the discussion (4.6)

Additional Comments: None

Overall Quality Determination

Medium

Page 121 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469417 Table: 2 of 6

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Zhang, S., Ireland, D., Sipes, N. S., Behl, M., Collins, E. S. (2019). Screening for neurotoxic potential of 15 flame retardants using freshwater planarians.

Neurotoxicology and Teratology 7354-66.

Overall Duration: 11-21 days; Exposure Duration: 11-21 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Invertebrate; Worms (e.g., Annelids, Nematodes); Dugesia japonica\ regenerative; Adult
Mortality

Tris(2-chloroethyl) phosphate (TCEP)

5469417

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1: Test Substance Identity
Metric 2: Test Substance Source

Metric 3: Test Substance Purity

High clearly identified with name and CAS #

Low Sourced from Sigma Aldrich, but chemical was not analytically verified by the labora-

tory.

High reported as 99.07% pure

Domain 2: Test Design

Metric 4:

Negative Controls

Low

Authors did not use a true negative control. "Negative" controls contained either ac-
etaminophen or L-ascorbic acid. A positive solvent control (0.5% DMSO) was also
used. The authors presented information from previous studies to verify that ac-
etaminophen or L-ascorbic acid did not affect the test organisms.

Metric 5:

Negative Control Response

High

100% survival in solvent control (0.5% DMSO), no mortality observed in "negative"
controls.

Metric 6:

Randomized Allocation

Low

no mention of randomization in this paper. Author did cite another paper which may
have described randomization

Domain 3: Exposure Characterization

Metric 7:

Experimental System/Test Media
Preparation

Low

minimal details provided making acceptability difficult to determine

Metric 8:

Consistency of Exposure
Administration

High

no inconsistencies were noticed or reported

Metric 9:

Measurement of Test Substance
Concentration

Low

exposure concentrations were not measured, high concentration listed as 99.5 uM in
supplemental data instead of a nominal value of 100 uM as stated in text

Metric 10:

Exposure Duration and Frequency

High

Exposure duration (12 days) was adequate to cause mortality for some of the chemicals
in this study (but not TCEP).

Metric 11:

Number of Exposure Groups/

High

Exposure concentrations seemed adequate to illicit a response (mortality) for most

Spacing of Exposure Levels

chemicals tested in this study. This was below the NOAEL for TCEP, but the purpose

of the study was to compare the mortality between chemicals within a specific group, so
this was not determined to affect the rating of this study.

Metric 12:

Testing at or Below Solubility Limit

High

Solvent (0.5% DMSO) was used to facilitate the dissolution of the test material into the
test medium.

Domain 4: Test Organism

Continued on next page .

Page 122 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469417 Table: 2 of 6

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Zhang, S., Ireland, D., Sipes, N. S., Behl, M., Collins, E. S. (2019). Screening for neurotoxic potential of 15 flame retardants using freshwater planarians.

Neurotoxicology and Teratology 7354-66.

Overall Duration: 11-21 days; Exposure Duration: 11-21 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Invertebrate; Worms (e.g., Annelids, Nematodes); Dugesia japonica\ regenerative; Adult
Mortality

Tris(2-chloroethyl) phosphate (TCEP)

5469417

Domain	Metric	Rating	Comments

Metric 13:

Test Organism Characteristics

High

Supplemental methods description describes the test organism in more detail (Zhang
et al., 2019). Test organisms were described as adults and "Test worms were manually
selected to fall within a certain range of sizes and we found full worm length, after au-
tomated size measurement, to be 7.3 6 2.3 mm (mean 6 SD), and tail worm length to be
7.3 mm 2.7 mm (mean 6 SD).

Metric 14:

Acclimatization and Pretreatment
Conditions

High

In supplemental materials, pretreatment procedure was described as: "Animals were
starved for at least 5 days before beingused for experiments and their containers were
cleaned immediately prior to worm selection for experiments." This was adequate for
gut-clearance.

Metric 15:

Number of Organisms and
Replicates per Group

Medium

8 individuals were used for each replicate.

Domain 5: Outcome Assessment







Metric 16:

Adequacy of Test Conditions

Low

Test conditions were fully described in supplemental methods paper (Zhang et al.,
2019): "Wells of a 48- well plate with 200 ml of IO water." The authors noted that in
the present study, they updated the feeding procedure, but this was not described clearly,
"we fed planarians used in FR screen 2 commercial freeze-dried organic chicken liver
(Amazon, Seattle, WA) to better control food quality and thus minimize animal fitness
variability." It was unclear when this supplemental feeding occurred during the exposure
period or if the lack of feeding could have contributed to additional mortality in some
chemicals in the screen (No mortality was observed for TCEP, so this was not an issue
for this chemcial).

Metric 17:

Outcome Assessment Methodology

High

Outcome assessment was adequate to describe the comparative mortality of several
flame retardants following exposure to the test material.

Metric 18:

Consistency of Outcome

High

assessment methods seemed consistent across groups

Assessment.

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test
Design and Procedures

Metric 20: Outcomes Unrelated to Exposure

Low	The study did not provide enough information to allow a comparison of environmental

conditions or other non-treatment-related factors across study groups. Besides the feed-
ing procedure, which did not contribute to mortality for TCEP, there were no reported
differences among the study groups in environmental conditions or other factors that
could influence the outcome assessment.

Medium There was no information in the study to suggest differences among groups in animal
attrition or health outcomes unrelated to exposure (e.g., infection) that could influence
the outcome assessment.

Domain 7: Data Presentation and Analysis

Continued on next page ...

Page 123 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469417 Table: 2 of 6

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Zhang, S., Ireland, D., Sipes, N. S., Behl, M., Collins, E. S. (2019). Screening for neurotoxic potential of 15 flame retardants using freshwater planarians.

Neurotoxicology and Teratology 7354-66.

Overall Duration: 11-21 days; Exposure Duration: 11-21 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Invertebrate; Worms (e.g., Annelids, Nematodes); Dugesia japonica\ regenerative; Adult
Mortality

Tris(2-chloroethyl) phosphate (TCEP)

5469417

Domain

Metric

Rating

Comments

Metric 21:

Statistical Methods

Low

Metric 22:

Reporting of Data

Low

A large amount of data was presented in the supplemental file (Zhang et al., 2019):
https://doi.Org/10.1016/j.ntt.2019.03.003. Data were not clear and interpretation was
difficult. It appeared that no mortality was observed in the regenerating adults following
exposure to TCEP for 7-12 days, but this was not clearly discussed.

Metric 23:

Explanation of Unexpected Outcomes

High

Additional Comments: None

Overall Quality Determination

Medium

Page 124 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469417 Table: 3 of 6

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Zhang, S., Ireland, D., Sipes, N. S., Behl, M., Collins, E. S. (2019). Screening for neurotoxic potential of 15 flame retardants using freshwater planarians.

Neurotoxicology and Teratology 7354-66.

Overall Duration: 11-21 days; Exposure Duration: 11-21 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Invertebrate; Worms (e.g., Annelids, Nematodes); Dugesia japonica\ regenerative; Adult

Development/Growth

Tris(2-chloroethyl) phosphate (TCEP)

5469417

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1: Test Substance Identity
Metric 2: Test Substance Source

Metric 3: Test Substance Purity

High clearly identified with name and CAS #

Low Sourced from Sigma Aldrich, but chemical was not analytically verified by the labora-

tory.

High reported as 99.07% pure

Domain 2: Test Design

Metric 4: Negative Controls

Metric 5: Negative Control Response
Metric 6: Randomized Allocation

Low Authors did not use a true negative control. "Negative" controls contained either ac-

High 100% survival in solvent control (0.5% DMSO), no mortality observed in "negative"

controls

Low no mention of randomization in this paper. Author did cite another paper which may

have described randomization

Domain 3: Exposure Characterization

Metric 7: Experimental System/Test Media
Preparation

Metric 8: Consistency of Exposure

Administration
Metric 9: Measurement of Test Substance

Concentration
Metric 10: Exposure Duration and Frequency

Metric 11: Number of Exposure Groups/
Spacing of Exposure Levels

Metric 12: Testing at or Below Solubility Limit

Low The experimental system and methods for preparation of test media were described in

High no inconsistencies were noticed or reported

Low Exposure concentrations were not measured, highest concentration listed as 99.5 uM in

supplemental data instead of a nominal value of 100 uM as stated in text

High Exposure duration (12 days) was adequate to elicit a response for the chemicals in this

study (but not TCEP).

High Exposure concentrations seemed adequate to illicit a response (development) for most

High Solvent (0.5% DMSO) was used to facilitate the dissolution of the test material into the

test medium.

Domain 4: Test Organism

Continued on next page .

Page 125 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469417 Table: 3 of 6

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Zhang, S., Ireland, D., Sipes, N. S., Behl, M., Collins, E. S. (2019). Screening for neurotoxic potential of 15 flame retardants using freshwater planarians.

Neurotoxicology and Teratology 7354-66.

Overall Duration: 11-21 days; Exposure Duration: 11-21 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Invertebrate; Worms (e.g., Annelids, Nematodes); Dugesia japonica\ regenerative; Adult

Development/Growth

Tris(2-chloroethyl) phosphate (TCEP)

5469417

Domain

Metric

Rating

Comments

Metric 13:

Test Organism Characteristics

High

Supplemental methods description describes the test organism in more detail (Zhang
et al., 2019). Test organisms were described as adults and "Test worms were manually
selected to fall within a certain range of sizes and we found full worm length, after au-
tomated size measurement, to be 7.3 6 2.3 mm (mean 6 SD), and tail worm length to be
7.3 mm 2.7 mm (mean 6 SD).

Metric 14:

Acclimatization and Pretreatment
Conditions

High

In supplemental materials, pretreatment procedure was described as: "Animals were
starved for at least 5 days before beingused for experiments and their containers were
cleaned immediately prior to worm selection for experiments." This was adequate for
gut-clearance.

Metric 15:

Number of Organisms and
Replicates per Group

Medium

8 individuals were used for each replicate.

Domain 5: Outcome Assessment







Metric 16:

Adequacy of Test Conditions

Medium

Test conditions were fully described in supplemental methods paper (Zhang et al.,
2019): "Wells of a 48- well plate with 200 ml of IO water." The authors noted that in
the present study, they updated the feeding procedure, but this was not described clearly,
"we fed planarians used in FR screen 2 commercial freeze-dried organic chicken liver
(Amazon, Seattle, WA) to better control food quality and thus minimize animal fitness
variability." It was unclear when this supplemental feeding occurred during the exposure
period or if the lack of feeding could have contributed to additional mortality in some
chemicals in the screen (No effects on development were observed for TCEP, so this was
not an issue for this chemcial).

Metric 17:

Outcome Assessment Methodology

High

outcome assessment was adequate, TCEP was essentially inactive for this test

Metric 18:

Consistency of Outcome
Assessment

High

assessment methods seemed consistent across groups

Domain 6: Confounding / Variable Control





Metric 19:

Confounding Variables in Test

Low

The study did not provide enough information to allow a comparison of environmental



Design and Procedures



conditions or other non-treatment-related factors across study groups. Besides the feed-





ing procedure, which did not contribute to mortality for TCEP, there were no reported
differences among the study groups in environmental conditions or other factors that
could influence the outcome assessment.

Metric 20:

Outcomes Unrelated to Exposure

High

There was no information in the study to suggest differences among groups in animal
attrition or health outcomes unrelated to exposure (e.g., infection) that could influence
the outcome assessment.

Domain 7: Data Presentation and Analysis

Continued on next page .

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469417 Table: 3 of 6

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Zhang, S., Ireland, D., Sipes, N. S., Behl, M., Collins, E. S. (2019). Screening for neurotoxic potential of 15 flame retardants using freshwater planarians.

Neurotoxicology and Teratology 7354-66.

Overall Duration: 11-21 days; Exposure Duration: 11-21 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Invertebrate; Worms (e.g., Annelids, Nematodes); Dugesia japonica\ regenerative; Adult

Development/Growth

Tris(2-chloroethyl) phosphate (TCEP)

5469417

Domain

Metric

Rating

Comments

Metric 21:

Statistical Methods

Low

Metric 22:

Reporting of Data

Low

A large amount of data was presented in the supplemental file (Zhang et al., 2019):
https://doi.Org/10.1016/j.ntt.2019.03.003. Data were not clear and interpretation was dif-
ficult. It appeared that no effect on development were observed in the adults following
exposure to TCEP for 7-12 days, but this was not clearly discussed.

Metric 23:

Explanation of Unexpected Outcomes

High

No effects on development were observed for entire test duration in any treatment for
TCEP. The lack of bioactivity observed in this study for TCEP was apparently not con-
sistent with previous studies showing bioactivity in Zebrafish. This was explained in the
discussion (4.6)

Additional Comments: None

Overall Quality Determination

Medium

Page 127 of 170

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Environmental Hazard Evaluation

HERO ID: 5469417 Table: 4 of 6

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Zhang, S., Ireland, D., Sipes, N. S., Behl, M., Collins, E. S. (2019). Screening for neurotoxic potential of 15 flame retardants using freshwater planarians.

Neurotoxicology and Teratology 7354-66.

Overall Duration: 11-21 days; Exposure Duration: 11-21 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Invertebrate; Worms (e.g., Annelids, Nematodes); Dugesia japonica\ regenerative; Adult
Behavioral

Tris(2-chloroethyl) phosphate (TCEP)

5469417

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1: Test Substance Identity
Metric 2: Test Substance Source

Metric 3: Test Substance Purity

High clearly identified with name and CAS #

Low Sourced from Sigma Aldrich, but chemical was not analytically verified by the labora-

tory.

High reported as 99.07% pure

Domain 2: Test Design

Metric 4: Negative Controls

Metric 5: Negative Control Response
Metric 6: Randomized Allocation

Low

High
Low

No effects observed in "negative" or solvent controls

No mention of randomization in this paper. Author did cite another paper which may
have described randomization.

Domain 3: Exposure Characterization
Metric 7:

Metric 8:
Metric 9:
Metric 10:
Metric 11:

Experimental System/Test Media
Preparation

Consistency of Exposure
Administration

Measurement of Test Substance
Concentration

Exposure Duration and Frequency

Number of Exposure Groups/
Spacing of Exposure Levels

Metric 12: Testing at or Below Solubility Limit

Low The experimental system and methods for preparation of test media were described in

High no inconsistencies were noticed or reported

Low exposure concentrations were not measured, high concentration listed as 99.5 uM in

supplemental data instead of a nominal value of 100 uM as stated in text

High Exposure duration (12 days) was adequate to to illicit a response for some of the chemi-

cals in this study (but not TCEP).

High Exposure concentrations seemed adequate to illicit a response (behavior) for most chem-

icals tested in this study. This was below the NOAEL for TCEP, but the purpose of the
study was to compare the effects on behavior between chemicals within a specific group,
so this was not determined to affect the rating of this study.

High Solvent (0.5% DMSO) was used to facilitate the dissolution of the test material into the

test medium.

Domain 4: Test Organism

Continued on next page .

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469417 Table: 4 of 6

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Zhang, S., Ireland, D., Sipes, N. S., Behl, M., Collins, E. S. (2019). Screening for neurotoxic potential of 15 flame retardants using freshwater planarians.

Neurotoxicology and Teratology 7354-66.

Overall Duration: 11-21 days; Exposure Duration: 11-21 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Invertebrate; Worms (e.g., Annelids, Nematodes); Dugesia japonica\ regenerative; Adult
Behavioral

Tris(2-chloroethyl) phosphate (TCEP)

5469417

Domain	Metric	Rating	Comments

Metric 13:

Test Organism Characteristics

High

Supplemental methods description describes the test organism in more detail (Zhang
et al., 2019). Test organisms were described as adults and "Test worms were manually
selected to fall within a certain range of sizes and we found full worm length, after au-
tomated size measurement, to be 7.3 6 2.3 mm (mean 6 SD), and tail worm length to be
7.3 mm 2.7 mm (mean 6 SD).

Metric 14:

Acclimatization and Pretreatment
Conditions

High

In supplemental materials, pretreatment procedure was described as: "Animals were
starved for at least 5 days before beingused for experiments and their containers were
cleaned immediately prior to worm selection for experiments." This was adequate for
gut-clearance.

Metric 15:

Number of Organisms and
Replicates per Group

Medium

8 individuals were used for each replicate.

Domain 5: Outcome Assessment







Metric 16:

Adequacy of Test Conditions

Low

Test conditions were fully described in supplemental methods paper (Zhang et al.,
2019): "Wells of a 48- well plate with 200 ml of IO water." The authors noted that in
the present study, they updated the feeding procedure, but this was not described clearly,
"we fed planarians used in FR screen 2 commercial freeze-dried organic chicken liver
(Amazon, Seattle, WA) to better control food quality and thus minimize animal fitness
variability." It was unclear when this supplemental feeding occurred during the exposure
period or if the lack of feeding could have contributed to additional mortality in some
chemicals in the screen (No mortality was observed for TCEP, so this was not an issue
for this Chemical).

Metric 17:

Outcome Assessment Methodology

High

Outcome assessment was adequate to describe the comparative mortality of several
flame retardants following exposure to the test material.

Metric 18:

Consistency of Outcome

High

assessment methods seemed consistent across groups

Assessment.

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test
Design and Procedures

Metric 20: Outcomes Unrelated to Exposure

Low	The study did not provide enough information to allow a comparison of environmental

conditions or other non-treatment-related factors across study groups. Besides the feed-
ing procedure, which did not contribute to mortality for TCEP, there were no reported
differences among the study groups in environmental conditions or other factors that
could influence the outcome assessment.

Medium There was no information in the study to suggest differences among groups in animal
attrition or health outcomes unrelated to exposure (e.g., infection) that could influence
the outcome assessment.

Domain 7: Data Presentation and Analysis

Continued on next page ...

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469417 Table: 4 of 6

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Zhang, S., Ireland, D., Sipes, N. S., Behl, M., Collins, E. S. (2019). Screening for neurotoxic potential of 15 flame retardants using freshwater planarians.

Neurotoxicology and Teratology 7354-66.

Overall Duration: 11-21 days; Exposure Duration: 11-21 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Invertebrate; Worms (e.g., Annelids, Nematodes); Dugesia japonica\ regenerative; Adult
Behavioral

Tris(2-chloroethyl) phosphate (TCEP)

5469417

Domain

Metric

Rating

Comments

Metric 21:

Statistical Methods

Low

Metric 22:

Reporting of Data

Low

A large amount of data was presented in the supplemental file (Zhang et al., 2019):
https://doi.Org/10.1016/j.ntt.2019.03.003. Data were not clear and interpretation was
difficult. It appeared that no behavioral effects were observed in the regenerating worms
following exposure to TCEP for 7-12 days, but this was not clearly discussed or ade-
quate to characterize a dose response.

Metric 23:

Explanation of Unexpected Outcomes

High

no unexpected outcomes for entire test duration in any treatment

Additional Comments: None

Overall Quality Determination

Medium

Page 130 of 170

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469417 Table: 5 of 6

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Zhang, S., Ireland, D., Sipes, N. S., Behl, M., Collins, E. S. (2019). Screening for neurotoxic potential of 15 flame retardants using freshwater planarians.

Neurotoxicology and Teratology 7354-66.

Overall Duration: 11-21 days; Exposure Duration: 11-21 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Invertebrate; Worms (e.g., Annelids, Nematodes); Dugesia japonica\ Adult
Other (please specify below) (AChE)

Tris(2-chloroethyl) phosphate (TCEP)

5469417

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1: Test Substance Identity
Metric 2: Test Substance Source

Metric 3: Test Substance Purity

High clearly identified with name and CAS #

Low Sourced from Sigma Aldrich, but chemical was not analytically verified by the labora-

tory.

High reported as 99.07% pure

Domain 2: Test Design

Metric 4:

Negative Controls

Low

Authors did not use a true negative control. A positive solvent control (0.5% DMSO)

was used.

Metric 5:

Negative Control Response

High

No effects on ACHE activity was reported in the solvent control

Metric 6:

Randomized Allocation

Low

No mention of randomization in this paper. Author did cite another paper which may

have described randomization.

Domain 3: Exposure Characterization

Metric 7:

Experimental System/Test Media

Low

The experimental system and methods for preparation of test media were described in

Preparation

adequate detail and appropriately accounted for the physical-chemical properties of the

test substance (solvent used due to the low solubility of the test material). Measured test

concentrations were not provided.

Metric 8:

Consistency of Exposure

High

no inconsistencies were noticed or reported

Administration

Metric 9:

Measurement of Test Substance

Low

Exposure concentrations were not measured, as this was conducted as a limit test, only

Concentration

one test concentration with a nominal value of 100 uM was used.

Metric 10:

Exposure Duration and Frequency

High

Exposure duration (12 days) was adequate to elicit significant loss of cholinesterase

activity for the other chemical in this study, EHDP, but not TCEP.

Metric 11:

Number of Exposure Groups/

N/A

As this was conducted as a limit test, only one concentration was used and no effects

Spacing of Exposure Levels

were observed.

Metric 12:

Testing at or Below Solubility Limit

High

Solvent (0.5% DMSO) was used to facilitate the dissolution of the test material into the

test medium.

Domain 4: Test Organism

Metric 13:

Test Organism Characteristics

High

Supplemental methods description describes the test organism in more detail (Zhang

et al., 2019). Test organisms were described as adults and "Test worms were manually

selected to fall within a certain range of sizes and we found full worm length, after au-

tomated size measurement, to be 7.3 6 2.3 mm (mean 6 SD), and tail worm length to be

7.3 mm 7- 2.7 mm (mean 6 SD).

Continued on next

page...

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December 2023

Tris(2-chloroethyl) phosphate (TCEP)	Environmental Hazard Evaluation	HERO ID: 5469417 Table: 5 of 6

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Zhang, S., Ireland, D., Sipes, N. S., Behl, M., Collins, E. S. (2019). Screening for neurotoxic potential of 15 flame retardants using freshwater planarians.

Neurotoxicology and Teratology 7354-66.

Overall Duration: 11-21 days; Exposure Duration: 11-21 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Invertebrate; Worms (e.g., Annelids, Nematodes); Dugesia japonica\ Adult
Other (please specify below) (AChE)

Tris(2-chloroethyl) phosphate (TCEP)

5469417

Domain	Metric	Rating	Comments

Metric 14:

Acclimatization and Pretreatment
Conditions

High

In supplemental materials, pretreatment procedure was described as: "Animals were
starved for at least 5 days before beingused for experiments and their containers were
cleaned immediately prior to worm selection for experiments." This was adequate for
gut-clearance.

Metric 15:

Number of Organisms and
Replicates per Group

Low

8 individuals were used for each replicate.

Domain 5: Outcome Assessment







Metric 16:

Adequacy of Test Conditions

Medium

Test conditions were fully described in supplemental methods paper (Zhang et al.,
2019): "Wells of a 48- well plate with 200 ml of IO water." The authors noted that in
the present study, they updated the feeding procedure, but this was not described clearly,
"we fed planarians used in FR screen 2 commercial freeze-dried organic chicken liver
(Amazon, Seattle, WA) to better control food quality and thus minimize animal fitness
variability." It was unclear when this supplemental feeding occurred during the exposure
period or if the lack of feeding could have contributed to additional effects in the screen
(No effects were observed following exposure to TCEP, so this was not an issue for this
chemical).

Metric 17:

Outcome Assessment Methodology

High

Outcome assessment was adequate to describe the comparative loss of cholinesterase
activity following exposure to two flame retardants, TCEP and EHDP.

Metric 18:

Consistency of Outcome

High

assessment methods seemed consistent across groups

Assessment.

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test
Design and Procedures

Metric 20: Outcomes Unrelated to Exposure

Low	The study did not provide enough information to allow a comparison of environmental

conditions or other non-treatment-related factors across study groups. Besides the feed-
ing procedure, which did not contribute to mortality for TCEP, there were no reported
differences among the study groups in environmental conditions or other factors that
could influence the outcome assessment.

Medium There was no information in the study to suggest differences among groups in animal
attrition or health outcomes unrelated to exposure (e.g., infection) that could influence
the outcome assessment.

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods
Metric 22: Reporting of Data

N/A	statistical analysis not applied as this was a limit test

Low	Data were not clear and interpretation was difficult. It appeared that no loss of

cholinesterase activity in the adults following exposure to TCEP for 7-12 days, but this
was not clearly discussed and raw data were not available.

Continued on next page ...

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469417 Table: 5 of 6

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Zhang, S., Ireland, D., Sipes, N. S., Behl, M., Collins, E. S. (2019). Screening for neurotoxic potential of 15 flame retardants using freshwater planarians.

Neurotoxicology and Teratology 7354-66.

Overall Duration: 11-21 days; Exposure Duration: 11-21 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Invertebrate; Worms (e.g., Annelids, Nematodes); Dugesia japonica\ Adult
Other (please specify below) (AChE)

Tris(2-chloroethyl) phosphate (TCEP)

5469417

Domain Metric

Rating

Comments

Metric 23: Explanation of Unexpected Outcomes

High

No mortalities for entire test duration in any treatment. The lack of bioactivity observed
in this study for TCEP was not consistent with previous studies showing bioactivity
in Zebrafish. This was explained in the discussion (4.6) as being related to the loss of
cholinesterase activity.

Additional Comments: None

Overall Quality Determination

Medium





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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469417 Table: 6 of 6

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Zhang, S., Ireland, D., Sipes, N. S., Behl, M., Collins, E. S. (2019). Screening for neurotoxic potential of 15 flame retardants using freshwater planarians.

Neurotoxicology and Teratology 7354-66.

Overall Duration: 11-21 days; Exposure Duration: 11-21 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Invertebrate; Worms (e.g., Annelids, Nematodes); Dugesia japonica\ Adult
Behavioral

Tris(2-chloroethyl) phosphate (TCEP)

5469417

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1: Test Substance Identity
Metric 2: Test Substance Source

Metric 3: Test Substance Purity

High clearly identified with name and CAS #

Low Sourced from Sigma Aldrich, but chemical was not analytically verified by the labora-

tory.

High reported as 99.07% pure

Domain 2: Test Design

Metric 4: Negative Controls

Metric 5: Negative Control Response
Metric 6: Randomized Allocation

Low

High
Low

No effects observed in "negative" or solvent controls

no mention of randomization in this paper. Author did cite another paper which may
have described randomization

Domain 3: Exposure Characterization
Metric 7:

Metric 8:
Metric 9:
Metric 10:

Experimental System/Test Media
Preparation

Consistency of Exposure
Administration

Measurement of Test Substance
Concentration

Exposure Duration and Frequency

Metric 11: Number of Exposure Groups/
Spacing of Exposure Levels

Metric 12: Testing at or Below Solubility Limit

Low The experimental system and methods for preparation of test media were described in

High no inconsistencies were noticed or reported

Low Exposure concentrations were not measured, high concentration listed as 99.5 uM in

supplemental data instead of a nominal value of 100 uM as stated in text

High Exposure duration (12 days) was adequate to to illicit a response for some of the chem-

icals in this study (but not TCEP, as no activity was observed up to the highest test con-
centration).

High Exposure concentrations seemed adequate to illicit a response (behavior) for most chem-

High Solvent (0.5% DMSO) was used to facilitate the dissolution of the test material into the

test medium.

Domain 4: Test Organism

Continued on next page .

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469417 Table: 6 of 6

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Zhang, S„ Ireland, D„ Sipes, N, S„ Behl, M„ Collins, E, S, (2019), Screening for neurotoxic potential of 15 flame retardants using freshwater planarians.

Neurotoxicology and Teratology 7354-66,

Overall Duration: 11-21 days; Exposure Duration: 11-21 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Invertebrate; Worms (e.g.. Annelids, Nematodes); Dugesia japonica; Adult
Behavioral

Tris(2-chloroethyl) phosphate (TCEP)

5469417

Domain	Metric	Rating	Comments

Metric 13;

Test Organism Characteristics

High

Supplemental methods description describes the test organism in more detail (Zhang
et al., 2019). Test organisms were described as adults and "Test worms were manually
selected to fall within a certain range of sizes and we found full worm length, after au-
tomated size measurement, to be 7.3 6 2.3 mm (mean 6 SD), and tail worm length to be
7.3 mm 2.7 mm (mean 6 SD).

Metric 14;

Acclimatization and Pretreatment
Conditions

High

In supplemental materials, pretreatment procedure was described as: "Animals were
starved for at least 5 days before beingused for experiments and their containers were
cleaned immediately prior to worm selection for experiments." This was adequate for
gut-clearance.

Metric 15;

Number of Organisms and
Replicates per Group

Medium

8 individuals were used for each replicate.

Domain 5; Outcome Assessment







Metric 16;

Adequacy of Test Conditions

Low

Test conditions were fully described in supplemental methods paper (Zhang et al.,
2019): "Wells of a 48- well plate with 200 ml of IO water." The authors noted that in
the present study, they updated the feeding procedure, but this was not described clearly,
"we fed planarians used in FR screen 2 commercial freeze-dried organic chicken liver
(Amazon, Seattle, WA) to better control food quality and thus minimize animal fitness
variability." It was unclear when this supplemental feeding occurred during the exposure
period or if the lack of feeding could have contributed to additional mortality in some
chemicals in the screen (No mortality was observed for TCEP, so this was not an issue
for this Chemical).

Metric 17;

Outcome Assessment Methodology

Medium

Outcome assessment was adequate to describe the comparative behavioral changes
following exposure to the test material and other flame retardants.

Metric 18;

Consistency of Outcome

High

assessment methods seemed consistent across groups

Assessment.

Domain 6; Confounding / Variable Control

Metric 19; Confounding Variables in Test
Design and Procedures

Metric 20; Outcomes Unrelated to Exposure

High	The study did not provide enough information to allow a comparison of environmental

conditions or other non-treatment-related factors across study groups. Besides the feed-
ing procedure, which did not contribute to mortality for TCEP, there were no reported
differences among the study groups in environmental conditions or other factors that
could influence the outcome assessment.

Medium There was no information in the study to suggest differences among groups in animal
attrition or health outcomes unrelated to exposure (e.g., infection) that could influence
the outcome assessment.

Domain 7; Data Presentation and Analysis

Continued on next page ...

Page 135 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469417 Table: 6 of 6

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Zhang, S., Ireland, D., Sipes, N. S., Behl, M., Collins, E. S. (2019). Screening for neurotoxic potential of 15 flame retardants using freshwater planarians.

Neurotoxicology and Teratology 7354-66.

Overall Duration: 11-21 days; Exposure Duration: 11-21 days

Aquatic (freshwater); Water; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Invertebrate; Worms (e.g., Annelids, Nematodes); Dugesia japonica\ Adult
Behavioral

Tris(2-chloroethyl) phosphate (TCEP)

5469417

Domain

Metric

Rating

Comments

Metric 21:

Statistical Methods

High

Metric 22:

Reporting of Data

High

A large amount of data was presented in the supplemental file (Zhang et al., 2019):
https://doi.Org/10.1016/j.ntt.2019.03.003. Data were not clear and interpretation was
difficult. It appeared that no behavioral effects were observed in the regenerating worms
following exposure to TCEP for 7-12 days, but this was not clearly discussed or ade-
quate to characterize a dose response.v

Metric 23:

Explanation of Unexpected Outcomes

High

no unexpected outcomes for entire test duration in any treatment

Additional Comments: None

Overall Quality Determination

Medium

Page 136 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5353113 Table: 1 of 3

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Fernie, K. J., Palace, V., Peters, L. E., Basu, N., Letcher, R. J., Karouna-Renier, N. K., Schultz, S. L., Lazarus, R. S., Rattner, B. A. (2015). Investigating
endocrine and physiological parameters of captive American kestrels exposed by diet to selected organophosphate flame retardants. Environmental Science
and Technology 49(12):7448-7455.

Overall Duration: > 21 days; Exposure Duration: 11-21 days
Terrestrial; Food/Diet; Dietary

Vertebrate; Avian; Falco sparverius\ Adult
Hepatic/Liver

Tris(2-chloroethyl) phosphate (TCEP)

5353113

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

identified in text and abstract

Metric 2:

Test Substance Source

Low

TCEP was purchased from Sigma-Aldrich but not analytically verified

Metric 3:

Test Substance Purity

High

purity reported as >97%

Domain 2: Test Design

Metric 4:

Negative Controls

High

Negative control of safflower oil (the delivery medium) was used.

Metric 5:

Negative Control Response

High

No reported mortalities in the negative controls and significant differences in the effects

were observed between the controls and exposure groups.

Metric 6:

Randomized Allocation

Low

random dosing was not reported

Domain 3: Exposure Characterization

Metric 7:

Experimental System/Test Media

High

Exposure preparation was well documented. Each kestrel ate one cockerel per day that

Preparation

had been injected with 50 "L of a safflower oil solution containing TCEP. Dosage was

calculated by weight to ensure consistent weight between the flame retardants used int

he study.

Metric 8:

Consistency of Exposure

High

No inconsistencies were reported

Administration

Metric 9:

Measurement of Test Substance

High

actual measured concentrations were reported

Concentration

Metric 10:

Exposure Duration and Frequency

High

exposure duration (21 days) was long enough to assess outcomes

Metric 11:

Number of Exposure Groups/

N/A

Authors used only one test concentration, normalized by body weight, across multiple

Spacing of Exposure Levels

chemicals so dosage spacing is not relevant in this case

Metric 12:

Testing at or Below Solubility Limit

N/A

dietary dose was administered via vegetable oil carrier, therefore the solubility was not

relevant

Domain 4: Test Organism

Metric

13:

Test Organism Characteristics

High

organisms were adequately described and appropriate

Metric

14:

Acclimatization and Pretreatment
Conditions

Low

no description of acclimatization process was provided

Metric

15:

Number of Organisms and
Replicates per Group

Low

7 individuals for each chemical with no replication. Given that the intention was to char-
acterize the comparative effects of the test material and not to calculate a dose response,

this is sufficient for statistical analysis
Continued on next page ...

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5353113 Table: 1 of 3

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Overall Duration: > 21 days; Exposure Duration: 11-21 days
Terrestrial; Food/Diet; Dietary

Vertebrate; Avian; Falco sparverius\ Adult
Hepatic/Liver

Tris(2-chloroethyl) phosphate (TCEP)

5353113

Domain

Metric

Rating

Comments

Domain 5: Outcome Assessment

Metric 16:
Metric 17:
Metric 18:

Adequacy of Test Conditions
Outcome Assessment Methodology
Consistency of Outcome
Assessment.

High test conditions were adequate and reasonably well documented
High methods were adequate to assess hepatic and thyroid hormone levels
High no inconsistencies were reported

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure

High no confounding variables were reported
High no unrelated outcomes were reported

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods

Metric 22: Reporting of Data

Metric 23: Explanation of Unexpected Outcomes

Low End points were compared for each treatment to vehicle-treated controls by parametric

analysis of variance (ANOVA). Calculations were not provided
High data was reported adequately and clearly
High no unexpected outcomes were reported

Additional Comments: None

Overall Quality Determination

High

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5353113 Table: 2 of 3

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Fernie, K. J., Palace, V., Peters, L. E., Basu, N., Letcher, R. J., Karouna-Renier, N. K., Schultz, S. L., Lazarus, R. S., Rattner, B. A. (2015). Investigating
endocrine and physiological parameters of captive American kestrels exposed by diet to selected organophosphate flame retardants. Environmental Science
and Technology 49(12):7448-7455.

Overall Duration: > 21 days; Exposure Duration: 11-21 days
Terrestrial; Food/Diet; Dietary

Vertebrate; Avian; Falco sparverius\ Adult
Other (please specify below) (Thyroid Function)

Tris(2-chloroethyl) phosphate (TCEP)

5353113

Domain

Metric

Rating

Comments

Domain 1: Test Substance







Metric 1:

Test Substance Identity

High

identified in text and abstract

Metric 2:

Test Substance Source

Low

TCEP was purchased from Sigma-Aldrich but not analytically verified

Metric 3:

Test Substance Purity

High

purity reported as >97%

Domain 2: Test Design







Metric 4:

Negative Controls

High

negative control of safflower oil (the delivery medium) was used

Metric 5:

Negative Control Response

High

No reported mortalities in the negative controls and significant differences in the ef-







fects were observed between the concentrations of FT3 and FT4 thyroid hormones and







thyroid gland histology between the control and exposure group of TCEP

Metric 6:

Randomized Allocation

Low

random dosing was not reported

Domain 3: Exposure Characterization







Metric 7:

Experimental System/Test Media

High

exposure preparation was well documented. Each kestrel ate one cockerel per day that



Preparation



had been injected with 50 "L of a safflower oil solution containing TCEP. Dosage was





calculated by weight to ensure consistent weight between the flame retardants used int







he study

Metric 8:

Consistency of Exposure

High

no inconsistencies were reported



Administration





Metric 9:

Measurement of Test Substance

High

actual measured concentrations were reported



Concentration





Metric 10:

Exposure Duration and Frequency

High

exposure duration (21 days) was long enough to assess outcomes

Metric 11:

Number of Exposure Groups/

N/A

authors used only one test concentration, normalized by body weight, across multiple



Spacing of Exposure Levels



chemicals so dosage spacing is not relevant in this case

Metric 12:

Testing at or Below Solubility Limit

N/A

dietary dose was administered via vegetable oil carrier, therefore the solubility was not







relevant.

Domain 4: Test Organism







Metric 13:

Test Organism Characteristics

High

organisms were adequately described and appropriate

Metric 14:

Acclimatization and Pretreatment

Low

no description of acclimatization process was provided



Conditions





Metric 15:

Number of Organisms and

Low

7 individuals for each chemical with no replication. Given that the intention was to char-



Replicates per Group



acterize the comparative effects of the test material and not to calculate a dose response,

this is sufficient for statistical analysis

Continued on next page ...

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5353113 Table: 2 of 3

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Overall Duration: > 21 days; Exposure Duration: 11-21 days
Terrestrial; Food/Diet; Dietary

Vertebrate; Avian; Falco sparverius\ Adult
Other (please specify below) (Thyroid Function)

Tris(2-chloroethyl) phosphate (TCEP)

5353113

Domain

Metric

Rating

Comments

Domain 5: Outcome Assessment

Metric 16:
Metric 17:

Metric 18:

Adequacy of Test Conditions
Outcome Assessment Methodology

Consistency of Outcome
Assessment.

High test conditions were adequate and reasonably well documented

High methods were adequate to characterize the comparative toxicity of several related chemi-
cals at the same exposure concentration
High no inconsistencies were reported

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure

High no confounding variables were reported
High no unrelated outcomes were reported

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods

Metric 22: Reporting of Data

Metric 23: Explanation of Unexpected Outcomes

Low

High
High

differences between the chemicals were compared for each treatment to vehicle-treated
controls by repeated measures (RM) analysis of variance (ANOVA). Calculations were
not provided.

data was reported adequately

no effects were not unexpected, explanation was adequate

Additional Comments: None

Overall Quality Determination

High

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December 2023

Environmental Hazard Evaluation

HERO ID: 5353113 Table: 3 of 3

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Overall Duration: > 21 days; Exposure Duration: 11-21 days
Terrestrial; Food/Diet; Dietary

Vertebrate; Avian; Falco sparverius\ Adult
ADME (biotransformation)

Tris(2-chloroethyl) phosphate (TCEP)

5353113

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

identified in text and abstract

Metric 2:

Test Substance Source

Low

TCEP was purchased from Sigma-Aldrich but not analytically verified

Metric 3:

Test Substance Purity

High

purity reported as >97%

Domain 2: Test Design

Metric 4:

Negative Controls

High

negative control of safflower oil (the delivery medium) was used

Metric 5:

Negative Control Response

High

No reported mortalities in the negative controls

Metric 6:

Randomized Allocation

Low

Random dosing was not reported

Domain 3: Exposure Characterization

Metric 7:

Experimental System/Test Media

High

exposure preparation was well documented. Each kestrel ate one cockerel per day that

Preparation

had been injected with 50 "L of a safflower oil solution containing TCEP. Dosage was

calculated by weight to ensure consistent weight between the flame retardants used int

he study.

Metric 8:

Consistency of Exposure

High

no inconsistencies were reported

Administration

Metric 9:

Measurement of Test Substance

High

actual measured concentrations were reported

Concentration

Metric 10:

Exposure Duration and Frequency

High

exposure duration (21 days) was long enough to assess outcomes

Metric 11:

Number of Exposure Groups/

N/A

authors used only one test concentration, normalized by body weight, across multiple

Spacing of Exposure Levels

chemicals so dosage spacing is not relevant in this case

Metric 12:

Testing at or Below Solubility Limit

N/A

Dietary dose was administered via vegetable oil carrier, therefore the solubility was not

relevant.

Domain 4: Test Organism

Metric 13:

Test Organism Characteristics

High

organisms were adequately described and appropriate

Metric 14:

Acclimatization and Pretreatment

Low

no description of acclimatization process was provided

Conditions

Metric 15:

Number of Organisms and

Low

7 individuals for each chemical with no replication. Given that the intention was to char-

Replicates per Group

acterize the comparative effects of the test material and not to calculate a dose response,

this is sufficient for statistical analysis

Domain 5: Outcome Assessment

Metric 16:

Adequacy of Test Conditions

High

test conditions were adequate and reasonably well documented

Continued on next page ...

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December 2023

Environmental Hazard Evaluation

HERO ID: 5353113 Table: 3 of 3

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Overall Duration: > 21 days; Exposure Duration: 11-21 days
Terrestrial; Food/Diet; Dietary

Vertebrate; Avian; Falco sparverius\ Adult
ADME (biotransformation)

Tris(2-chloroethyl) phosphate (TCEP)

5353113

Domain

Metric

Rating

Comments

Metric 17:
Metric 18:

Outcome Assessment Methodology

Consistency of Outcome
Assessment

High
High

methods were adequate to characterize the comparative toxicity of several related chemi-
cals at the same exposure concentration
no inconsistencies were reported

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure

High
High

no confounding variables were reported
no unrelated outcomes were reported

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods

Metric 22: Reporting of Data

Metric 23: Explanation of Unexpected Outcomes

Low

High
High

effects were compared for each treatment to vehicle-treated controls by nonparametric
analysis of variance (ANOVA) and Repeated Measures ANOVA on ranked data using
SAS 9.3. Calculations were not provided,
data was reported adequately

low TCEP levels in organisms was given possible explanations

Additional Comments: None

Overall Quality Determination

High

Page 142 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5165206 Table: 1 of 1

Study Citation:
Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Stauffer Chem Co, (1981). Toxicology reports on FYROL FR-2 (volume I - II) with attachments and cover letter dated 020381. 8100271#88-8100271.
Overall Duration: > 21 days; Exposure Duration: 4 - 10 days
Terrestrial; Food/Diet; Dietary

Vertebrate; Avian; Gallus gallus domesticus\ Adult
Behavioral

Tris(2-chloroethyl) phosphate (TCEP)

5165206

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1: Test Substance Identity

Metric 2: Test Substance Source
Metric 3: Test Substance Purity

Uninformative

High
Low

Fyrol CEF. Lot f 3587-2-1, was identified as the test chemical with no other identifiers
(e.g., CASRN, structure, formula). Online search shows FYROL CEF as a synonym for
Tris(2-chloroethyl) phosphate CAS 119-96-8. However, Fyrol CEF is also listed as a
synonym for at least one other CASRN.

Source reported as Specialty Chemical Division
Purity not reported.

Domain 2: Test Design

Metric 4: Negative Controls
Metric 5: Negative Control Response
Metric 6: Randomized Allocation

High
High
Low

negative and positive controls used.

Negative response for positive and negative controls.

Allocation method not reported.

Domain 3: Exposure Characterization
Metric 7:

Metric 8:

Metric 9:

Metric 10:
Metric 11:

Metric 12:

Experimental System/Test Media Low

Preparation

Consistency of Exposure High

Administration

Measurement of Test Substance Medium

Concentration

Exposure Duration and Frequency High

Number of Exposure Groups/ Uninformative

Spacing of Exposure Levels

Testing at or Below Solubility Limit N/A

Reported details were limited

Exposure administration consistent across groups.

Method of measuring exposure concentration not reported.

Exposure duration was adequate for the test.

Only a single exposure level for test with no response.

Dosage administered orally.

Domain 4: Test Organism

Metric 13:
Metric 14:

Metric 15:

Test Organism Characteristics
Acclimatization and Pretreatment
Conditions

Number of Organisms and
Replicates per Group

Low Only age of chickens reported.

Low Acclimation or pretreatment not reported.

Medium Number of organism per group and replicates were appropriate for test.

Domain 5: Outcome Assessment

Metric 16:
Metric 17:

Adequacy of Test Conditions
Outcome Assessment Methodology

High
Low

Environmental conditions not reported.
Response to treatment was zero.

Continued on next page .

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5165206 Table: 1 of 1

... continued from previous page

Study Citation:
Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Stauffer Chem Co, (1981). Toxicology reports on FYROL FR-2 (volume I - II) with attachments and cover letter dated 020381. 8100271#88-8100271.
Overall Duration: > 21 days; Exposure Duration: 4 - 10 days
Terrestrial; Food/Diet; Dietary

Vertebrate; Avian; Gallus gallus domesticus\ Adult
Behavioral

Tris(2-chloroethyl) phosphate (TCEP)

5165206

Domain

Metric

Rating

Comments

Metric 18:

Consistency of Outcome
Assessment

High

Outcome assessments were consistent.

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure

High
Medium

No confounding variables were reported,
no differences were reported.

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods

Metric 22: Reporting of Data

Metric 23: Explanation of Unexpected Outcomes

Low
High
High

Qualitative score for behavior were summed.
All outcomes reported,
no unexpected outcomes reported.

Additional Comments: None

Overall Quality Determination

Uninformative

Page 144 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 3479540 Table: 1 of 1

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Overall Duration: 0 - 4 days (0-96h); Exposure Duration: 0 - 4 days (0-96h)

Terrestrial; Food/Diet; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Invertebrate; Worms (e.g., Annelids, Nematodes); Caenorhabditis elegans\ Bristol N2; Larvae

Development/Growth

Tris(2-chloroethyl) phosphate (TCEP)

3479540

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1: Test Substance Identity
Metric 2: Test Substance Source

Metric 3: Test Substance Purity

High Test substance was identified by name and CAS #

Low Test substance source was reported as Sigma-Aldrich, but it did not appear to be ana-

lyzed by the performing laboratory
High Test substance purity was 98.95%

Domain 2: Test Design

Metric 4: Negative Controls
Metric 5: Negative Control Response

Metric 6: Randomized Allocation

High All treated groups were compared to a DMSO vehicle controls.

High The biological response of the negative control was reported and adequate. Untreated

nematodes reached L4 stage in growth.

Low Researchers did not report how organisms were allocated to study groups.

Domain 3: Exposure Characterization
Metric 7:

Metric 8:

Metric 9:

Metric 10:
Metric 11:

Metric 12:

Experimental System/Test Media
Preparation

Consistency of Exposure
Administration

Measurement of Test Substance
Concentration

Exposure Duration and Frequency
Number of Exposure Groups/
Spacing of Exposure Levels
Testing at or Below Solubility Limit

Low DMSO solvent was used in the preparation of the test media stock solutions, but little

other information was provided on preparation. The experimental system was 96 well
plates.

High Details of exposure administration were reported and exposures were administered

consistently across study groups. 48h exposure in 96 well plates with 50 nematodes per
well.

Low Exposure concentrations were not measured or measurements were not reported.

High The duration of exposure was reported and suitable for the study type-48h exposure.

High 12 exposure groups were reported with a concentration range from luM to 200uM.

Found in the supplementary material.

High DMSO solvent concentration was appropriate at 0.1% (v/v)

Domain 4: Test Organism

Metric

13:

Test Organism Characteristics

Metric

14:

Acclimatization and Pretreatment

Conditions

Metric

15:

Number of Organisms and

Replicates per Group

High The test organisms were adequately described and were obtained from a reliable source

Low The study did not report whether test organisms were acclimatized

Medium The numbers of test organisms and replicates were reported and sufficient to character-
ize toxicological effect. 50 nematodes per well with at least 3 replicates of each concen-
tration according to the supplementary material.

Continued on next page ...

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 3479540 Table: 1 of 1

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Overall Duration: 0 - 4 days (0-96h); Exposure Duration: 0 - 4 days (0-96h)

Terrestrial; Food/Diet; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Invertebrate; Worms (e.g., Annelids, Nematodes); Caenorhabditis elegans\ Bristol N2; Larvae

Development/Growth

Tris(2-chloroethyl) phosphate (TCEP)

3479540

Domain

Metric

Rating

Comments

Domain 5: Outcome Assessment

Metric 16: Adequacy of Test Conditions

Metric 17: Outcome Assessment Methodology

Metric 18: Consistency of Outcome
Assessment

Low Boyd et al 2009 was cited for organism maintenance. Kept at 20 C and fed E.coli, other-

wise little other housing information was provided.

Medium Rice et al 2014 was cited for assessment methodology. Some details were provided in

the paper- organisms' gross morphology and developmental stage were assessed as well
as size

High Organisms were assessed 48h after exposure using gross morphology and developmental

stage. Biosort was used to assess size and number of organisms

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure

Low The study did not provide enough information to allow a comparison of environmental

conditions or other non-treatment-related factors across study groups.

Medium There was no information in the study to suggest differences among groups in animal
attrition or health outcomes unrelated to exposure

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods
Metric 22: Reporting of Data

Metric 23: Explanation of Unexpected Outcomes

High
High

High

Statistical methods were adequately described

Data for exposure-related findings were presented for each treatment and control group
and were adequate to determine values for the endpoint of interest. Data was provided in
the supplementary material. Control response was described in the text.

There were no unexpected outcomes

Additional Comments: None

Overall Quality Determination

Medium

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 3975281 Table: 1 of 3

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Behl, M., Rice, J. R., Smith, M. V., Co, C. A., Bridge, M. F., Hsieh, J. H., Freedman, J. H., Boyd, W. A. (2016). Editor's highlight: Comparative toxicity
of organophosphate flame retardants and polybrominated diphenyl ethers to Caenorhabditis elegans. Toxicological Sciences 154(2):241-252.

Overall Duration: 0 - 4 days (0-96h); Exposure Duration: 0 - 4 days (0-96h)

Terrestrial; Cell Culture Media; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Invertebrate; Worms (e.g., Annelids, Nematodes); Caenorhabditis elegans\ Bristol N2 (wild-type); Larvae

Reproductive/Teratogenic

Tris(2-chloroethyl) phosphate (TCEP)

3975281

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

TCEP (tris(2-chloroethyl)

Metric 2:

Test Substance Source

High

Purchased from Sigma Aldrich Chemical Co

Metric 3:

Test Substance Purity

High

98.95% Purity

Domain 2: Test Design

Metric 4:

Negative Controls

High

Authors only reported two positive control groups used, containing chlorpyrifos and

"aromatic OPFR TOCP".

Metric 5:

Negative Control Response

High

No toxicity response from the positive control groups used.

Metric 6:

Randomized Allocation

Low

How organisms were allocation was not reported.

Domain 3: Exposure Characterization

Metric 7:

Experimental System/Test Media

Medium

Study did not comment on whether agar plates or well-plates were sealed.

Preparation

Metric 8:

Consistency of Exposure

High

Exposure administration was consistent for all groups.

Administration

Metric 9:

Measurement of Test Substance

Medium

Exposure concentrations were measured at beginning of exposure, but not at the end of

Concentration

the exposure period.

Metric 10:

Exposure Duration and Frequency

High

Exposure duration and frequency appropriate for test.

Metric 11:

Number of Exposure Groups/

Low

Number exposure groups and frequency were appropriate for other tested chemicals,

Spacing of Exposure Levels

however no adverse effects were reported for TCEP.

Metric 12:

Testing at or Below Solubility Limit

Low

Reporting were insufficient to determine if solubility limits were exceeded.

Domain 4: Test Organism

Metric 13:

Test Organism Characteristics

High

Test organisms were adequately described and source provided.

Metric 14:

Acclimatization and Pretreatment

High

pretreatments and acclimation periods were adequately described.

Conditions

Metric 15:

Number of Organisms and

Medium

Number of test organisms and replicates were adequate.

Replicates per Group

Domain 5: Outcome Assessment

Metric 16:

Adequacy of Test Conditions

High

Test organism environmental conditions were acceptable.

Metric 17:

Outcome Assessment Methodology

High

Intended outcomes reported.

Continued on next page ...

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 3975281 Table: 1 of 3

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Overall Duration: 0 - 4 days (0-96h); Exposure Duration: 0 - 4 days (0-96h)

Terrestrial; Cell Culture Media; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Invertebrate; Worms (e.g., Annelids, Nematodes); Caenorhabditis elegans\ Bristol N2 (wild-type); Larvae

Reproductive/Teratogenic

Tris(2-chloroethyl) phosphate (TCEP)

3975281

Domain

Metric

Rating

Comments

Metric 18:

Consistency of Outcome
Assessment

High

Assessments were consistent across groups

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure

High
Medium

No reported differences among groups.
No reported differences among groups.

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods
Metric 22: Reporting of Data

Metric 23: Explanation of Unexpected Outcomes

High
Low

Low

Statistical methods were clearly described.

Test results for exposure groups were reported in poorly rendered, graphs and only sum-
marized cursorily in the text. No adverse outcomes were reported.

No explanations was provided why TCEP was the only flame retardant that was inactive
across all of the C.elegans endpoints.

Additional Comments: None

Overall Quality Determination

High

Page 148 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 3975281 Table: 2 of 3

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Overall Duration: 4-10 days; Exposure Duration: 0 - 4 days (0-96h)

Terrestrial; Cell Culture Media; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Invertebrate; Worms (e.g., Annelids, Nematodes); Caenorhabditis elegans\ Bristol N2 (wild-type); Larvae
Development/Growth
Tris(2-chloroethyl) phosphate (TCEP)

3975281

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

TCEP (tris(2-chloroethyl)

Metric 2:

Test Substance Source

High

Purchased from Sigma Aldrich Chemical Co

Metric 3:

Test Substance Purity

High

98.95% Purity

Domain 2: Test Design

Metric 4:

Negative Controls

High

Authors only reported two positive control groups used, containing chlorpyrifos and

"aromatic OPFR TOCP".

Metric 5:

Negative Control Response

High

No toxicity response from the positive control groups used.

Metric 6:

Randomized Allocation

Low

How organisms were allocation was not reported.

Domain 3: Exposure Characterization

Metric 7:

Experimental System/Test Media

Medium

Study did not comment on whether agar plates or well-plates were sealed.

Preparation

Metric 8:

Consistency of Exposure

High

Exposure administration was consistent for all groups.

Administration

Metric 9:

Measurement of Test Substance

Medium

Exposure concentrations were measured at beginning of exposure, but not at the end of

Concentration

the exposure period.

Metric 10:

Exposure Duration and Frequency

High

Exposure duration and frequency appropriate for test.

Metric 11:

Number of Exposure Groups/

Low

Number exposure groups and frequency were appropriate for other tested chemicals,

Spacing of Exposure Levels

however no adverse effects were reported for TCEP.

Metric 12:

Testing at or Below Solubility Limit

Low

Reporting were insufficient to determine if solubility limits were exceeded.

Domain 4: Test Organism

Metric 13:

Test Organism Characteristics

High

Test organisms were adequately described and source provided.

Metric 14:

Acclimatization and Pretreatment

High

pretreatments and acclimation periods were adequately described.

Conditions

Metric 15:

Number of Organisms and

Medium

Number of test organisms and replicates were adequate.

Replicates per Group

Domain 5: Outcome Assessment

Metric 16:

Adequacy of Test Conditions

High

Test organism environmental conditions were acceptable.

Metric 17:

Outcome Assessment Methodology

High

Intended outcomes reported.

Metric 18:

Consistency of Outcome

High

Assessments were consistent across groups

Assessment.

Continued on next page ...

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 3975281 Table: 2 of 3

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Overall Duration: 4-10 days; Exposure Duration: 0 - 4 days (0-96h)

Terrestrial; Cell Culture Media; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Invertebrate; Worms (e.g., Annelids, Nematodes); Caenorhabditis elegans\ Bristol N2 (wild-type); Larvae
Development/Growth
Tris(2-chloroethyl) phosphate (TCEP)

3975281

Domain Metric

Rating

Comments

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test

High

No reported differences among groups.

Design and Procedures

Metric 20: Outcomes Unrelated to Exposure

Medium

No reported differences among groups.

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods
Metric 22: Reporting of Data

Metric 23: Explanation of Unexpected Outcomes

High Statistical methods were clearly described.

Low Test results for exposure groups were reported in poorly rendered, graphs and only sum-
marized cursorily in the text. No adverse outcomes were reported.

Low No explanations was provided why TCEP was the only flame retardant that was inactive
across all of the C.elegans endpoints.

Additional Comments: None

Overall Quality Determination

High

Page 150 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 3975281 Table: 3 of 3

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Overall Duration: 4-10 days; Exposure Duration: 0 - 4 days (0-96h)

Terrestrial; Cell Culture Media; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Invertebrate; Worms (e.g., Annelids, Nematodes); Caenorhabditis elegans\ Bristol N2 (wild-type); Larvae
Behavioral

Tris(2-chloroethyl) phosphate (TCEP)

3975281

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

TCEP (tris(2-chloroethyl)

Metric 2:

Test Substance Source

High

Purchased from Sigma Aldrich Chemical Co

Metric 3:

Test Substance Purity

High

98.95% Purity

Domain 2: Test Design

Metric 4:

Negative Controls

High

Authors only reported two positive control groups used, containing chlorpyrifos and

"aromatic OPFR TOCP".

Metric 5:

Negative Control Response

High

No toxicity response from the positive control groups used.

Metric 6:

Randomized Allocation

Low

How organisms were allocation was not reported.

Domain 3: Exposure Characterization

Metric 7:

Experimental System/Test Media

Medium

Study did not comment on whether agar plates or well-plates were sealed.

Preparation

Metric 8:

Consistency of Exposure

High

Exposure administration was consistent for all groups.

Administration

Metric 9:

Measurement of Test Substance

Medium

Exposure concentrations were measured at beginning of exposure, but not at the end of

Concentration

the exposure period.

Metric 10:

Exposure Duration and Frequency

High

Exposure duration and frequency appropriate for test.

Metric 11:

Number of Exposure Groups/

Low

Number exposure groups and frequency were appropriate for other tested chemicals,

Spacing of Exposure Levels

however no adverse effects were reported for TCEP.

Metric 12:

Testing at or Below Solubility Limit

Low

Reporting were insufficient to determine if solubility limits were exceeded.

Domain 4: Test Organism

Metric 13:

Test Organism Characteristics

High

Test organisms were adequately described and source provided.

Metric 14:

Acclimatization and Pretreatment

High

pretreatments and acclimation periods were adequately described.

Conditions

Metric 15:

Number of Organisms and

Medium

Number of test organisms and replicates were adequate.

Replicates per Group

Domain 5: Outcome Assessment

Metric 16:

Adequacy of Test Conditions

High

Test organism environmental conditions were acceptable.

Metric 17:

Outcome Assessment Methodology

High

Intended outcomes reported.

Metric 18:

Consistency of Outcome

High

Assessments were consistent across groups

Assessment.

Continued on next page ...

Page 151 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 3975281 Table: 3 of 3

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Overall Duration: 4-10 days; Exposure Duration: 0 - 4 days (0-96h)

Terrestrial; Cell Culture Media; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Invertebrate; Worms (e.g., Annelids, Nematodes); Caenorhabditis elegans\ Bristol N2 (wild-type); Larvae
Behavioral

Tris(2-chloroethyl) phosphate (TCEP)

3975281

Domain Metric

Rating

Comments

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test

High

No reported differences among groups.

Design and Procedures

Metric 20: Outcomes Unrelated to Exposure

Medium

No reported differences among groups.

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods
Metric 22: Reporting of Data

Metric 23: Explanation of Unexpected Outcomes

High Statistical methods were clearly described.

Low Test results for exposure groups were reported in poorly rendered, graphs and only sum-
marized cursorily in the text. No adverse outcomes were reported.

Low No explanations was provided why TCEP was the only flame retardant that was inactive
across all of the C.elegans endpoints.

Additional Comments: None

Overall Quality Determination

High

Page 152 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469475 Table: 1 of 5

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Xu, T., Li, P., Wu, S., Lei, L., He, D. (2017). Tris(2-chloroethyl) phosphate (TCEP) and tris(2-chloropropyl) phosphate (TCPP) induce locomotor deficits
and dopaminergic degeneration in Caenorhabditis elegans. Toxicology Research 6(l):63-72.

Overall Duration: 0 - 4 days (0-96h); Exposure Duration: 0 - 4 days (0-96h)

Terrestrial; Cell Culture Media; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Invertebrate; Worms (e.g., Annelids, Nematodes); Caenorhabditis elegans; Wild Type (Bristol, N2); Larvae
Development/Growth
Tris(2-chloroethyl) phosphate (TCEP)

5469475

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

Name and CASRN were reported.

Metric 2:

Test Substance Source

High

Sourced from Sigma Aldrich.

Metric 3:

Test Substance Purity

High

All chemicals were Analytical Grade.

Domain 2: Test Design

Metric 4:

Negative Controls

High

Control was K Medium as were the rest of the treatments.

Metric 5:

Negative Control Response

High

Body length of control reported in Figure 2, Al; seems appropriate for baseline as low

concentration TCEP treatments had similar body lengths.

Metric 6:

Randomized Allocation

Low

No mention of how organisms were allocated.

Domain 3: Exposure Characterization

Metric 7:

Experimental System/Test Media

High

"TCEP solutions were prepared in K-medium and the control groupis K-medium.

Preparation

Worms were exposed to a series of concentrations of TCEP (50, 250, 500, 750, 1000

mg in 24-well plates." Post exposure set up adequately described.

Metric 8:

Consistency of Exposure

Medium

Test organisms exposed for 3 days in 24-well plates. Lighting and other incubation

Administration

conditions not described.

Metric 9:

Measurement of Test Substance

Medium

Exposure concentrations were not measured but based on professional judgment of

Concentration

experimental design and nature of test substance, actual concentrations are likely to be

similar to nominal concentrations. These minor uncertainties or limitations are unlikely

to have a substantial impact on results.

Metric 10:

Exposure Duration and Frequency

Medium

Unclear if 3 day exposure duration with endpoints taken immediately post-exposure is

sufficient for determining impact on body length.

Metric 11:

Number of Exposure Groups/

High

Exposure covered a wide range with good spacing, 0-1000 mg/L.

Spacing of Exposure Levels

Metric 12:

Testing at or Below Solubility Limit

Medium

Sigma Aldrich states TCEP solubility in water is 50 mg/mL (50 g/L); 1000 mg/L (1 g/

L), the high dose, would be far under the solubility limit.

Domain 4: Test Organism

Metric 13:

Test Organism Characteristics

High

Good description of C. elegans and how test organisms were obtained. "All strains of

C. elegans were obtained from the Caenorhabditis Genetics Center (University of Min-

nesota, Minneapolis, MN, USA), and maintained in terms of standard protocols as previ-

ously described."

Metric 14:

Acclimatization and Pretreatment

Low

Authors did not state if organisms were acclimated to 24-well plates prior to chemical

Conditions

exposure.

Continued on next

page...

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469475 Table: 1 of 5

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Xu, T., Li, P., Wu, S., Lei, L., He, D. (2017). Tris(2-chloroethyl) phosphate (TCEP) and tris(2-chloropropyl) phosphate (TCPP) induce locomotor deficits
and dopaminergic degeneration in Caenorhabditis elegans. Toxicology Research 6(l):63-72.

Overall Duration: 0 - 4 days (0-96h); Exposure Duration: 0 - 4 days (0-96h)

Terrestrial; Cell Culture Media; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Invertebrate; Worms (e.g., Annelids, Nematodes); Caenorhabditis elegans\ Wild Type (Bristol, N2); Larvae
Development/Growth
Tris(2-chloroethyl) phosphate (TCEP)

5469475

Domain

Metric

Rating

Comments

Metric 15:

Number of Organisms and
Replicates per Group

Medium

Sufficient to establish an adequate response. 50-100 worms per exposure concentration,
experiments performed in quadruplet.

Domain 5: Outcome Assessment

Metric 16:

Metric 17:
Metric 18:

Adequacy of Test Conditions
Outcome Assessment Methodology

Consistency of Outcome
Assessment

Medium
High

High

Details on total volume per well (assuming 500 uL) as well as temperature and lighting
during exposure in 24-well plates lacking.

Authors reported how lengths were assessed: "After being exposed to TCEP (0, 50, 250,
500, 750, 1000 mg/L)...for 3 d, worms were washed three times with M9 buffer, and
then transferred to agar-padded slides and sealed with a coverslip which were immobi-
lized with 100 mM sodium azide. Then body lengths of nematodes were measured using
an imaging system."

No mention of any inconsistencies.

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure

High
High

No reported differences of any variables that would alter the outcome.
No unrelated issues were reported.

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods

Metric 22: Reporting of Data

Metric 23: Explanation of Unexpected Outcomes

High

High
High

"All data were expressed as mean ± Standard Deviation (SD). Mean differences be-
tween treated groups and controls were determined by one-way analysis of variance
(ANOVA), followed by Dunnett"s test. A p-value of less than 0.05 was considered sig-
nificant."

Data for control and treatment groups were reported graphically with standard deviation

(Figure 2, Al) and in text form.

No unexpected outcomes, deviations were reasonable.

Additional Comments: The form 3 for biomarker endpoint applies to GFP expression in BZ555 and YFP expression in NL5901 strains. The form 3 for neurological endpoint
applies to both local movement and locomotor assays. Well reported study.

Overall Quality Determination

High

Page 154 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469475 Table: 2 of 5

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Overall Duration: > 21 days; Exposure Duration: 0 - 4 days (0-96h)

Terrestrial; Cell Culture Media; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Invertebrate; Worms (e.g., Annelids, Nematodes); Caenorhabditis elegans; Wild Type (Bristol, N2); Larvae
Mortality

Tris(2-chloroethyl) phosphate (TCEP)

5469475

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

Name and CASRN were reported.

Metric 2:

Test Substance Source

High

Sourced from Sigma Aldrich.

Metric 3:

Test Substance Purity

High

Percent purity not reported however chemicals stated to be Analytical Grade.

Domain 2: Test Design

Metric 4: Negative Controls
Metric 5: Negative Control Response

Metric 6: Randomized Allocation

High Control was K Medium as were the rest of the treatments.

Medium Control response of near 100% survival, from graph. However, it was unclear if this
response was scaled to 100% of control survival.

Low No mention of how organisms were allocated.

Domain 3: Exposure Characterization

Metric 7:

Experimental System/Test Media

High

"TCEP solutions were prepared in K-medium and the control groupis K-medium.

Preparation

Worms were exposed to a series of concentrations of TCEP (50, 250, 500, 750, 1000

mg in 24-well plates." Post exposure set up adequately described.

Metric 8:

Consistency of Exposure

Medium

Nothing to suggest exposure was inconsistent across groups.

Administration

Metric 9:

Measurement of Test Substance

Medium

Concentrations not measured but no indication that measured concentrations should

Concentration

deviate from nominal concentrations.

Metric 10:

Exposure Duration and Frequency

High

Sufficient duration (3 days) to obtain mortality over time post-exposure.

Metric 11:

Number of Exposure Groups/

High

Exposure covered a wide range with good spacing, 0-1000 mg/L.

Spacing of Exposure Levels

Metric 12:

Testing at or Below Solubility Limit

High

Sigma Aldrich states TCEP solubility in water is 50 mg/mL (50 g/L); 1000 mg/L (1 g/

L), the high dose, would be far under the solubility limit.

Domain 4: Test Organism

Metric 13: Test Organism Characteristics

Metric 14: Acclimatization and Pretreatment

Conditions
Metric 15: Number of Organisms and
Replicates per Group

High Good description of C. elegans and how test organisms were obtained. "All strains of

C. elegans were obtained from the Caenorhabditis Genetics Center (University of Min-
nesota, Minneapolis, MN, USA), and maintained in terms of standard protocols as previ-
ously described."

Low Authors did not state if test organisms were acclimated to 24-well plates prior to chemi-

cal exposure.

Medium Sufficient to establish an adequate response. 50-100 worms per exposure concentration,
experiments performed in quadruplet.

Domain 5: Outcome Assessment

Continued on next page ...

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469475 Table: 2 of 5

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Xu, T., Li, P., Wu, S., Lei, L., He, D. (2017). Tris(2-chloroethyl) phosphate (TCEP) and tris(2-chloropropyl) phosphate (TCPP) induce locomotor deficits
and dopaminergic degeneration in Caenorhabditis elegans. Toxicology Research 6(1 ):63-72.

Overall Duration: > 21 days; Exposure Duration: 0 - 4 days (0-96h)

Terrestrial; Cell Culture Media; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Invertebrate; Worms (e.g., Annelids, Nematodes); Caenorhabditis elegans\ Wild Type (Bristol, N2); Larvae
Mortality

Tris(2-chloroethyl) phosphate (TCEP)

5469475

Domain

Metric

Rating

Comments

Metric 16:
Metric 17:
Metric 18:

Adequacy of Test Conditions
Outcome Assessment Methodology
Consistency of Outcome
Assessment

Low
High
High

No details on physical conditions of test organisms

Reported how mortalities were assessed (every 2 days) over time post exposure.
No mention of any inconsistencies.

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure

High
High

No mention of any variables that would alter the outcome.
No unrelated issues were reported.

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods

Metric 22: Reporting of Data

Metric 23: Explanation of Unexpected Outcomes

High

High
High

Percentages were reported as well as a mention of significant difference. Statistics for
lifespan assay sufficiently described in cited methods Xu et al 2016 as well as under
'Statistical Analysis' section.

Data for control and treatment groups were reported graphically (Figure 2, CI) and in
text form.

No unexpected outcomes, deviations were reasonable.

Additional Comments: The form 3 for biomarker endpoint applies to GFP expression in BZ555 and YFP expression in NL5901 strains. The form 3 for neurological endpoint
applies to both local movement and locomotor assays. Well reported study.

Overall Quality Determination

Medium

Page 156 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469475 Table: 3 of 5

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Overall Duration: 0 - 4 days (0-96h); Exposure Duration: 0 - 4 days (0-96h)

Terrestrial; Cell Culture Media; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Invertebrate; Worms (e.g., Annelids, Nematodes); Caenorhabditis elegans; BZ555; Larvae
Mechanistic-Biomarkers (exposure and effect)

Tris(2-chloroethyl) phosphate (TCEP)

5469475

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1:
Metric 2:
Metric 3:

Test Substance Identity
Test Substance Source
Test Substance Purity

High
High
High

Name and CASRN were reported.

Sourced from Sigma Aldrich.

All chemicals were Analytical Grade.

Domain 2: Test Design

Metric 4:
Metric 5:
Metric 6:

Negative Controls
Negative Control Response
Randomized Allocation

High
High
Low

Control was K Medium as were the rest of the treatments.

Control responses shown in Figure 6G and 7F and seem reasonable.
No mention of how organisms were allocated.

Domain 3: Exposure Characterization
Metric 7:

Metric 8:
Metric 9:

Metric 10:

Experimental System/Test Media
Preparation

Consistency of Exposure
Administration

Measurement of Test Substance
Concentration

Exposure Duration and Frequency

High

Medium
Medium

High

"TCEP solutions were prepared in K-medium and the control groupis K-medium.

Worms were exposed to a series of concentrations of TCEP (50, 250, 500, 750, 1000
mg in 24-well plates." Post exposure set up adequately described.

Test organisms exposed for 3 days in 24-well plates. Lighting and other incubation
conditions not described.

Exposure concentrations were not measured but based on professional judgment of
experimental design and nature of test substance, actual concentrations are likely to be
similar to nominal concentrations. These minor uncertainties or limitations are unlikely
to have a substantial impact on results.

3 day exposure period should be sufficient for GFP and YFP expression in the transgenic
strains.

Exposure covered a wide range with good spacing, 0-1000 mg/L.

Sigma Aldrich states TCEP solubility in water is 50 mg/mL (50 g/L); 1000 mg/L (1 g/
L), the high dose, would be far under the solubility limit.

Metric 11:
Metric 12:

Number of Exposure Groups/
Spacing of Exposure Levels
Testing at or Below Solubility Limit

High
High

Domain 4: Test Organism

Metric 13:

Metric 14:
Metric 15:

Test Organism Characteristics

Acclimatization and Pretreatment
Conditions

Number of Organisms and
Replicates per Group

High

Medium
Medium

Good description of C. elegans and how test organisms were obtained. "All strains of
C. elegans were obtained from the Caenorhabditis Genetics Center (University of Min-
nesota, Minneapolis, MN, USA), and maintained in terms of standard protocols as previ-
ously described."

Authors did not state whether organisms acclimated to 24-well plates prior to exposure.
At least 30 nematodes per group analyzed for GFP expression or YFP expression.

Continued on next

page...

Page 157 of 170

-------
Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469475 Table: 3 of 5

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Xu, T., Li, P., Wu, S., Lei, L., He, D. (2017). Tris(2-chloroethyl) phosphate (TCEP) and tris(2-chloropropyl) phosphate (TCPP) induce locomotor deficits
and dopaminergic degeneration in Caenorhabditis elegans. Toxicology Research 6(l):63-72.

Overall Duration: 0 - 4 days (0-96h); Exposure Duration: 0 - 4 days (0-96h)

Terrestrial; Cell Culture Media; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Invertebrate; Worms (e.g., Annelids, Nematodes); Caenorhabditis elegans\ BZ555; Larvae
Mechanistic-Biomarkers (exposure and effect)

Tris(2-chloroethyl) phosphate (TCEP)

5469475

Domain

Metric

Rating

Comments

Domain 5: Outcome Assessment

Metric 16:

Metric 17:

Adequacy of Test Conditions
Outcome Assessment Methodology

Metric 18: Consistency of Outcome
Assessment

Medium Details on total volume per well (assuming 500 uL) as well as temperature and lighting
during exposure in 24-well plates lacking.

High Analysis of PDat-1 in dopamine neurons via GFP tagging and alpha-synuclein in mus-
cles via YFP tagging adequately described. Established transgenic strains utilized for
these experiments.

High Outcome assessment methodology adequately described by authors as well as cited
references.

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test	High

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure	High

No mention of any variables that would alter the outcome.
No outcomes unrelated to exposure were reported.

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods

Metric 22: Reporting of Data

Metric 23: Explanation of Unexpected Outcomes

High "All data were expressed as mean ± Standard Deviation (SD).Mean differences between
treated groups and controls weredetermined by one-way analysis of variance (ANOVA),
followedby Dunnett"s test. A p-value of less than 0.05 was consideredsignificant."

High Data for GFP expression in dopamine neurons (in BZ555 strain) shown in Figure 6 and
data for YFP expression in muscles (in NL5901 strain) shown in Figure 7 as well as
described in the text.

High No unexpected outcomes, deviations were reasonable.

Additional Comments:

The form 3 for biomarker endpoint applies to GFP expression in BZ555 and YFP expression in NL5901 strains. The form 3 for neurological endpoint
applies to both local movement and locomotor assays. Well reported study.	

Overall Quality Determination

High

Page 158 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469475 Table: 4 of 5

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Overall Duration: 0 - 4 days (0-96h); Exposure Duration: 0 - 4 days (0-96h)

Terrestrial; Cell Culture Media; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Invertebrate; Worms (e.g., Annelids, Nematodes); Caenorhabditis elegans; Wild Type (Bristol, N2); Larvae
Neurological

Tris(2-chloroethyl) phosphate (TCEP)

5469475

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

Name and CASRN were reported.

Metric 2:

Test Substance Source

High

Sourced from Sigma Aldrich.

Metric 3:

Test Substance Purity

High

All chemicals were Analytical Grade.

Domain 2: Test Design

Metric 4:

Negative Controls

High

Control was K Medium as were the rest of the treatments.

Metric 5:

Negative Control Response

High

Control responses in local movement and locomotor assays shown in Figures 3, 4, and

5 seem reasonable (i.e. similar to responses shown in low TCEP concentration treat-

ments).

Metric 6:

Randomized Allocation

Low

No mention of how organisms were allocated.

Domain 3: Exposure Characterization

Metric 7:

Experimental System/Test Media

High

Test set up described in 24-well plates as well as in cited references for NGM plates

Preparation

(Tsalik and Hobert 2003; Donnelly et al 2013; Gallagher et al 2013)

Metric 8:

Consistency of Exposure

High

Test organisms exposed to TCEP 3 days prior to local movement and locomotor assays

Administration

as stated in Figure 3 and Figure 4.

Metric 9:

Measurement of Test Substance

Medium

Exposure concentrations were not measured but based on professional judgment of

Concentration

experimental design and nature of test substance, actual concentrations are likely to be

similar to nominal concentrations. These minor uncertainties or limitations are unlikely

to have a substantial impact on results.

Metric 10:

Exposure Duration and Frequency

High

Exposure duration was 3 days which should be sufficient for establishing effects on local

movement and locomotion.

Metric 11:

Number of Exposure Groups/

High

Exposure covered a wide range with good spacing, 0-1000 mg/L

Spacing of Exposure Levels

Metric 12:

Testing at or Below Solubility Limit

High

Sigma Aldrich states TCEP solubility in water is 50 mg/mL (50 g/L); 1000 mg/L (1 g/

L), the high dose, would be far under the solubility limit.

Domain 4: Test Organism

Metric 13:

Test Organism Characteristics

High

Good description of C. elegans and how test organisms were obtained. "All strains of

C. elegans were obtained from the Caenorhabditis Genetics Center (University of Min-

nesota, Minneapolis, MN, USA), and maintained in terms of standard protocols as previ-

ously described."

Metric 14:

Acclimatization and Pretreatment

Medium

Test organisms acclimated briefly before local movement and locomotor assays in NGM

Conditions

plates. Authors did not state whether test organisms were acclimated to 24-well plate

prior to chemical exposure.

Continued on next page ...

Page 159 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469475 Table: 4 of 5

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Overall Duration: 0 - 4 days (0-96h); Exposure Duration: 0 - 4 days (0-96h)

Terrestrial; Cell Culture Media; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Invertebrate; Worms (e.g., Annelids, Nematodes); Caenorhabditis elegans\ Wild Type (Bristol, N2); Larvae
Neurological

Tris(2-chloroethyl) phosphate (TCEP)

5469475

Domain

Metric

Rating

Comments

Metric 15:

Number of Organisms and
Replicates per Group

Medium

Sufficient to establish an adequate response. 50-100 worms per exposure concentration
in 24-well plates, experiments performed in quadruplet.

Domain 5: Outcome Assessment

Metric 16:

Metric 17:
Metric 18:

Adequacy of Test Conditions

Outcome Assessment Methodology

Consistency of Outcome
Assessment

Medium

High
High

Details on total volume per well (assuming 500 uL) as well as temperature and light-
ing during exposure in 24-well plates lacking. Conditions in NGM plates adequately
described by authors as well as cited references.

Authors adequately described how local movement and locomotion was assessed via
text and cited references.

No mention of any inconsistencies in any of the assessments.

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure

High
High

No mention of any variables that would alter the outcome.
No outcomes unrelated to exposure were reported.

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods

Metric 22: Reporting of Data

Metric 23: Explanation of Unexpected Outcomes

High

High
High

"All data were expressed as mean ± Standard Deviation (SD).Mean differences between
treated groups and controls weredetermined by one-way analysis of variance (ANOVA),
followedby Dunnett"s test. A p-value of less than 0.05 was consideredsignificant."

Data for local movement shown in Figure 3 and data for locomotion shown in Figures 4
and 5 as well as described in text.

No unexpected outcomes, deviations were reasonable.

Additional Comments: The form 3 for biomarker endpoint applies to GFP expression in BZ555 and YFP expression in NL5901 strains. The form 3 for neurological endpoint
applies to both local movement and locomotor assays. Well reported study.

Overall Quality Determination

High

Page 160 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469475 Table: 5 of 5

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Overall Duration: 4-10 days; Exposure Duration: 4-10 days

Terrestrial; Cell Culture Media; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Invertebrate; Worms (e.g., Annelids, Nematodes); Caenorhabditis elegans; Wild Type (Bristol, N2); Larvae
Mortality

Tris(2-chloroethyl) phosphate (TCEP)

5469475

Domain

Metric

Rating

Comments

Domain 1: Test Substance

Metric 1:

Test Substance Identity

High

Name and CASRN were reported.

Metric 2:

Test Substance Source

High

Sourced from Sigma Aldrich.

Metric 3:

Test Substance Purity

High

Percent purity not reported but all chemicals were Analytical Grade.

Domain 2: Test Design

Metric 4:

Negative Controls

High

Control was K Medium as were the rest of the treatments.

Metric 5:

Negative Control Response

Medium

Control response of near 100% survival, from graph; however, not clear if control sur-

vival was scaled to 100% for the purposes of the graph.

Metric 6:

Randomized Allocation

Low

No mention of how organisms were allocated.

Domain 3: Exposure Characterization

Metric 7:

Experimental System/Test Media

High

TCEP prepared in K medium, 24-well plate set up adequately described.

Preparation

Metric 8:

Consistency of Exposure

Medium

Nothing to suggest exposure was inconsistent across groups.

Administration

Metric 9:

Measurement of Test Substance

Medium

Exposure concentrations were not measured but based on professional judgment of

Concentration

experimental design and nature of test substance, actual concentrations are likely to be

similar to nominal concentrations. These minor uncertainties or limitations are unlikely

to have a substantial impact on results.

Metric 10:

Exposure Duration and Frequency

High

Sufficient duration to obtain a good dose response; 1,3,6 days.

Metric 11:

Number of Exposure Groups/

High

Exposure covered a wide range with good spacing, almost two orders of magnitude.

Spacing of Exposure Levels

Metric 12:

Testing at or Below Solubility Limit

High

Sigma Aldrich states TCEP solubility in water is 50 mg/mL (50 g/L); 1000 mg/L (1 g/

L), the high dose, would be far under the solubility limit.

Domain 4: Test Organism

Metric 13:

Test Organism Characteristics

High

Good description of C. elegans and how test organisms were obtained. "All strains of

C. elegans were obtained from the Caenorhabditis Genetics Center (University of Min-

nesota, Minneapolis, MN, USA), and maintained in terms of standard protocols as previ-

ously described."

Metric 14:

Acclimatization and Pretreatment

Medium

Unclear if organisms were acclimated to 24-well plates prior to treatment.

Conditions

Metric 15:

Number of Organisms and

Medium

Adequate for endpoint determinations.

Replicates per Group

Continued on next page ...

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469475 Table: 5 of 5

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Overall Duration: 4-10 days; Exposure Duration: 4-10 days

Terrestrial; Cell Culture Media; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Invertebrate; Worms (e.g., Annelids, Nematodes); Caenorhabditis elegans\ Wild Type (Bristol, N2); Larvae
Mortality

Tris(2-chloroethyl) phosphate (TCEP)

5469475

Domain

Metric

Rating

Comments

Domain 5: Outcome Assessment

Metric 16:

Metric 17:
Metric 18:

Adequacy of Test Conditions

Outcome Assessment Methodology
Consistency of Outcome
Assessment

Medium

High
High

Details on total volume per well (assuming 500 uL) as well as temperature and lighting
during exposure in 24-well plates lacking.

Reported how mortalities were assessed-unresponsive to gentle needle probe.
No mention of any inconsistencies.

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure

High
High

No mention of any variables that would alter the outcome.
No unrelated issues were reported.

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods

Metric 22: Reporting of Data

Metric 23: Explanation of Unexpected Outcomes

High

High
High

Confidence intervals were included with LC50 values. "The median lethal concentra-
tions (LC50) of TCEP and TCPP were determined bylinear regression analysis with
Graphpad Prism."

LC50 values for 1, 3, and 6 day lethality tests were reported in text and dose responses
were shown in graphically. Well documented.

No unexpected outcomes, deviations were reasonable.

Overall Quality Determination

High

Page 162 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469239 Table: 1 of 4

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Yang, Y., Xiao, Y., Chang, Y., Cui, Y., Klobucar, G., Li, M. (2018). Intestinal damage, neurotoxicity and biochemical responses caused by tris (2-
chloroethyl) phosphate and tricresyl phosphate on earthworm. Ecotoxicology and Environmental Safety 15878-86.

Overall Duration: 11-21 days; Exposure Duration: 11-21 days

Terrestrial; Soil; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Invertebrate; Worms (e.g., Annelids, Nematodes); Eiseniafetida\ Adult
Mortality

Tris(2-chloroethyl) phosphate (TCEP)

5469239

Domain

Metric

Rating

Comments

Domain 1: Test Substance







Metric 1:

Test Substance Identity

High

positively identified in abstract and main body

Metric 2:

Test Substance Source

Low

Obtained from TCI development Company, not analytically verified

Metric 3:

Test Substance Purity

High

percent purity reported as >97%

Domain 2: Test Design

Metric 4: Negative Controls
Metric 5: Negative Control Response

Metric 6: Randomized Allocation

High Control group was used, no chemical control was used

High Mortality was less than 5% across treatments. Treatments were significantly different

than controls.

Low no mention of random allocation

Domain 3: Exposure Characterization

Metric 7:

Experimental System/Test Media

High

well documented experimental set up



Preparation





Metric 8:

Consistency of Exposure

High

no inconsistencies were noted



Administration





Metric 9:

Measurement of Test Substance

Low

chemical treatment levels were not measured



Concentration





Metric 10:

Exposure Duration and Frequency

High

exposure duration followed guidelines and was sufficient to assess outcomes

Metric 11:

Number of Exposure Groups/

High

exposure concentration range was sufficient to assess outcomes



Spacing of Exposure Levels





Metric 12:

Testing at or Below Solubility Limit

Medium

no mention of how chemical dispersion occurred, this is a terrestrial study not sure if

solubility is an issue for this

Domain 4: Test Organism

Metric 13:
Metric 14:

Metric 15:

Test Organism Characteristics
Acclimatization and Pretreatment
Conditions

Number of Organisms and
Replicates per Group	

High well documented organism characteristics

High well documented organism acclimatization and pretreatment procedure
Medium organism numbers sufficient to assess outcome

Domain 5: Outcome Assessment

Metric 16:
Metric 17:

Adequacy of Test Conditions	High

Outcome Assessment Methodology Medium

well documented and adequate test conditions

subsampling periodically for mortality is not the recommended method

Continued on next page ...

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469239 Table: 1 of 4

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Yang, Y., Xiao, Y., Chang, Y., Cui, Y., Klobucar, G., Li, M. (2018). Intestinal damage, neurotoxicity and biochemical responses caused by tris (2-
chloroethyl) phosphate and tricresyl phosphate on earthworm. Ecotoxicology and Environmental Safety 15878-86.

Overall Duration: 11-21 days; Exposure Duration: 11-21 days

Terrestrial; Soil; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Invertebrate; Worms (e.g., Annelids, Nematodes); Eiseniafetida\ Adult
Mortality

Tris(2-chloroethyl) phosphate (TCEP)

5469239

Domain

Metric

Rating

Comments

Metric 18:

Consistency of Outcome
Assessment

High

no inconsistencies in the assessment were noted

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure

High
High

no confounding variables in the assessment were noted
no unrelated outcomes were noted in the assessment

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods

Metric 22: Reporting of Data

Metric 23: Explanation of Unexpected Outcomes

High
Low
High

Statistical methods were adequate and reported in section 2.8
no actual values for mortality were reported
no unexpected outcomes were reported

Additional Comments: None

Overall Quality Determination

High

Page 164 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469239 Table: 2 of 4

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Yang, Y., Xiao, Y., Chang, Y., Cui, Y., Klobucar, G., Li, M. (2018). Intestinal damage, neurotoxicity and biochemical responses caused by tris (2-
chloroethyl) phosphate and tricresyl phosphate on earthworm. Ecotoxicology and Environmental Safety 15878-86.

Overall Duration: 11-21 days; Exposure Duration: 11-21 days

Terrestrial; Soil; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Invertebrate; Worms (e.g., Annelids, Nematodes); Eiseniafetida\ Adult
Development/Growth
Tris(2-chloroethyl) phosphate (TCEP)

5469239

Domain

Metric

Rating

Comments

Domain 1: Test Substance







Metric 1:

Test Substance Identity

High

positively identified in abstract and main body

Metric 2:

Test Substance Source

Low

Obtained from TCI development Company, not analytically verified

Metric 3:

Test Substance Purity

High

percent purity reported as >97%

Domain 2: Test Design

Metric 4: Negative Controls
Metric 5: Negative Control Response

Metric 6: Randomized Allocation

High Control group was used. No chemical control was used

High Mortality was less than 5% across treatments. Treatments were significantly different

than controls.

Low no mention of random allocation

Domain 3: Exposure Characterization

Metric 7:

Experimental System/Test Media

High

well documented experimental set up



Preparation





Metric 8:

Consistency of Exposure

High

no inconsistencies were noted



Administration





Metric 9:

Measurement of Test Substance

Low

chemical treatment levels were not measured



Concentration





Metric 10:

Exposure Duration and Frequency

High

exposure duration followed guidelines and was sufficient to assess outcomes

Metric 11:

Number of Exposure Groups/

High

exposure concentration range was sufficient to assess outcomes



Spacing of Exposure Levels





Metric 12:

Testing at or Below Solubility Limit

Medium

no mention of how chemical dispersion occurred, this is a terrestrial study not sure if







solubility is an issue for this

Domain 4: Test Organism







Metric 13:

Test Organism Characteristics

High

well documented organism characteristics

Metric 14:

Acclimatization and Pretreatment

High

well documented organism acclimatization and pretreatment procedure



Conditions





Metric 15:

Number of Organisms and

Medium

organism numbers sufficient to assess outcome



Replicates per Group





Domain 5: Outcome Assessment







Metric 16:

Adequacy of Test Conditions

High

well documented and adequate test conditions

Metric 17:

Outcome Assessment Methodology

High

assessment methods were straightforward

Metric 18:

Consistency of Outcome

High

no inconsistencies in the assessment were noted



Assessment.





Continued on next page ...

Page 165 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469239 Table: 2 of 4

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Yang, Y., Xiao, Y., Chang, Y., Cui, Y., Klobucar, G., Li, M. (2018). Intestinal damage, neurotoxicity and biochemical responses caused by tris (2-
chloroethyl) phosphate and tricresyl phosphate on earthworm. Ecotoxicology and Environmental Safety 15878-86.

Overall Duration: 11-21 days; Exposure Duration: 11-21 days

Terrestrial; Soil; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Invertebrate; Worms (e.g., Annelids, Nematodes); Eiseniafetida\ Adult
Development/Growth
Tris(2-chloroethyl) phosphate (TCEP)

5469239

Domain

Metric

Rating

Comments

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure

High no confounding variables in the assessment were noted
High no unrelated outcomes were noted in the assessment

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods	High Statistical significance compared to control reported in Figure 1.

Metric 22: Reporting of Data	High growth rate values were reported reasonably well

Metric 23: Explanation of Unexpected Outcomes Medium a trend was reported but no explanation for a mid treatment level relative decrease
Additional Comments: None

Overall Quality Determination

High

Page 166 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469239 Table: 3 of 4

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Yang, Y., Xiao, Y., Chang, Y., Cui, Y., Klobucar, G., Li, M. (2018). Intestinal damage, neurotoxicity and biochemical responses caused by tris (2-
chloroethyl) phosphate and tricresyl phosphate on earthworm. Ecotoxicology and Environmental Safety 15878-86.

Overall Duration: 11-21 days; Exposure Duration: 11-21 days

Terrestrial; Soil; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Invertebrate; Worms (e.g., Annelids, Nematodes); Eiseniafetida\ Adult
Neurological

Tris(2-chloroethyl) phosphate (TCEP)

5469239

Domain

Metric

Rating

Comments

Domain 1: Test Substance







Metric 1:

Test Substance Identity

High

positively identified in abstract and main body

Metric 2:

Test Substance Source

Low

Obtained from TCI development Company, not analytically verified

Metric 3:

Test Substance Purity

High

percent purity reported as >97%

Domain 2: Test Design

Metric 4: Negative Controls
Metric 5: Negative Control Response

Metric 6: Randomized Allocation

High Control group was used. No chemical control was used

High Mortality was less than 5% across treatments. Treatments were significantly different

than controls.

Low no mention of random allocation

Domain 3: Exposure Characterization

Metric 7:

Experimental System/Test Media

High

well documented experimental set up



Preparation





Metric 8:

Consistency of Exposure

High

no inconsistencies were noted



Administration





Metric 9:

Measurement of Test Substance

Low

chemical treatment levels were not measured



Concentration





Metric 10:

Exposure Duration and Frequency

High

exposure duration followed guidelines and was sufficient to assess outcomes

Metric 11:

Number of Exposure Groups/

High

exposure concentration range was sufficient to assess outcomes



Spacing of Exposure Levels





Metric 12:

Testing at or Below Solubility Limit

Medium

no mention of how chemical dispersion occurred, this is a terrestrial study not sure if







solubility is an issue for this

Domain 4: Test Organism







Metric 13:

Test Organism Characteristics

High

well documented organism characteristics

Metric 14:

Acclimatization and Pretreatment

High

well documented organism acclimatization and pretreatment procedure



Conditions





Metric 15:

Number of Organisms and

Medium

organism numbers sufficient to assess outcome



Replicates per Group





Domain 5: Outcome Assessment







Metric 16:

Adequacy of Test Conditions

High

well documented and adequate test conditions

Metric 17:

Outcome Assessment Methodology

High

assessment methods were straightforward

Metric 18:

Consistency of Outcome

High

no inconsistencies in the assessment were noted



Assessment.





Continued on next page ...

Page 167 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469239 Table: 3 of 4

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Yang, Y., Xiao, Y., Chang, Y., Cui, Y., Klobucar, G., Li, M. (2018). Intestinal damage, neurotoxicity and biochemical responses caused by tris (2-
chloroethyl) phosphate and tricresyl phosphate on earthworm. Ecotoxicology and Environmental Safety 15878-86.

Overall Duration: 11-21 days; Exposure Duration: 11-21 days

Terrestrial; Soil; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Invertebrate; Worms (e.g., Annelids, Nematodes); Eiseniafetida\ Adult
Neurological

Tris(2-chloroethyl) phosphate (TCEP)

5469239

Domain

Metric

Rating

Comments

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure

High no confounding variables in the assessment were noted
High no unrelated outcomes were noted in the assessment

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods

Metric 22: Reporting of Data

Metric 23: Explanation of Unexpected Outcomes

High author noted what statistics were applied, gene transcription, GSH and AChE did not

follow a clear dose response
High Transcriptions, GSH levels and AChE levels rate values were reported reasonably well
High trends were reported ando explanations were provided for results not following trends

Additional Comments: None

Overall Quality Determination

High

Page 168 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469239 Table: 4 of 4

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Yang, Y., Xiao, Y., Chang, Y., Cui, Y., Klobucar, G., Li, M. (2018). Intestinal damage, neurotoxicity and biochemical responses caused by tris (2-
chloroethyl) phosphate and tricresyl phosphate on earthworm. Ecotoxicology and Environmental Safety 15878-86.

Overall Duration: 11-21 days; Exposure Duration: 11-21 days

Terrestrial; Soil; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Invertebrate; Worms (e.g., Annelids, Nematodes); Eiseniafetida\ Adult
Gastrointestinal

Tris(2-chloroethyl) phosphate (TCEP)

5469239

Domain

Metric

Rating

Comments

Domain 1: Test Substance







Metric 1:

Test Substance Identity

High

positively identified in abstract and main body

Metric 2:

Test Substance Source

Low

Obtained from TCI development Company, not analytically verified

Metric 3:

Test Substance Purity

High

percent purity reported as >97%

Domain 2: Test Design

Metric 4: Negative Controls
Metric 5: Negative Control Response

Metric 6: Randomized Allocation

High Control group was used. No chemical control was used

High Mortality was less than 5% across treatments. Treatments were significantly different

than controls.

Low no mention of random allocation

Domain 3: Exposure Characterization

Metric 7:

Experimental System/Test Media

High

well documented experimental set up



Preparation





Metric 8:

Consistency of Exposure

High

no inconsistencies were noted



Administration





Metric 9:

Measurement of Test Substance

Low

chemical treatment levels were not measured



Concentration





Metric 10:

Exposure Duration and Frequency

High

exposure duration followed guidelines and was sufficient to assess outcomes

Metric 11:

Number of Exposure Groups/

High

exposure concentration range was sufficient to assess outcomes



Spacing of Exposure Levels





Metric 12:

Testing at or Below Solubility Limit

Medium

no mention of how chemical dispersion occurred, this is a terrestrial study not sure if







solubility is an issue for this

Domain 4: Test Organism







Metric 13:

Test Organism Characteristics

High

well documented organism characteristics

Metric 14:

Acclimatization and Pretreatment

High

well documented organism acclimatization and pretreatment procedure



Conditions





Metric 15:

Number of Organisms and

Medium

organism numbers sufficient to assess outcome



Replicates per Group





Domain 5: Outcome Assessment







Metric 16:

Adequacy of Test Conditions

High

well documented and adequate test conditions

Metric 17:

Outcome Assessment Methodology

High

assessment methods were straightforward

Metric 18:

Consistency of Outcome

High

no inconsistencies in the assessment were noted



Assessment.





Continued on next page ...

Page 169 of 170

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Tris(2-chloroethyl) phosphate (TCEP)

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December 2023

Environmental Hazard Evaluation

HERO ID: 5469239 Table: 4 of 4

... continued from previous page

Study Citation:

Duration:
Exposure Route,
Media, Path:

Taxa, Species, Age:
Health Outcome:
Chemical:

HERO ID:

Yang, Y., Xiao, Y., Chang, Y., Cui, Y., Klobucar, G., Li, M. (2018). Intestinal damage, neurotoxicity and biochemical responses caused by tris (2-
chloroethyl) phosphate and tricresyl phosphate on earthworm. Ecotoxicology and Environmental Safety 15878-86.

Overall Duration: 11-21 days; Exposure Duration: 11-21 days

Terrestrial; Soil; Not determined by study authors (i.e., chemical of interest in exposure water, but unable to determine exact uptake route)

Invertebrate; Worms (e.g., Annelids, Nematodes); Eiseniafetida\ Adult
Gastrointestinal

Tris(2-chloroethyl) phosphate (TCEP)

5469239

Domain

Metric

Rating

Comments

Domain 6: Confounding / Variable Control

Metric 19: Confounding Variables in Test

Design and Procedures
Metric 20: Outcomes Unrelated to Exposure

High no confounding variables in the assessment were noted
High no unrelated outcomes were noted in the assessment

Domain 7: Data Presentation and Analysis

Metric 21: Statistical Methods

Metric 22: Reporting of Data

Metric 23: Explanation of Unexpected Outcomes

High author noted what statistics were applied, intestinal damage and DNA breaks followed a

reasonable dose response biochemical changes did not follow a clear dose response
High intestinal damage, DNA breaks and biochemical changes were reported reasonably well
High trends were reported and explanations were provided for results not following trends

Additional Comments: None

Overall Quality Determination

High

Page 170 of 170

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