Annex XV report

PROPOSAL FOR IDENTIFICATION OF A SUBSTANCE
OF VERY HIGH CONCERN ON THE BASIS OF THE
CRITERIA SET OUT IN REACH ARTICLE 57

Substance Name: Triphenyl phosphate (TPhP)EC Number: 204-112-2
CAS Number: 115-86-6

Submitted by: France
Date: 23 Feb 2024

ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHCThis document has been prepared according to template: TEM-0049.04

CONTENTSPROPOSAL FOR IDENTIFICATION OF A SUBSTANCE OF VERY HIGH CONCERN ON THE
BASIS OF THE CRITERIA SET OUT IN REACH ARTICLE 57 ........................................................... 8
PART I ...................................................................................................................................................................10JUSTIFICATION ................................................................................................................................................10
1.

IDENTITY OF THE SUBSTANCE AND PHYSICAL AND CHEMICAL PROPERTIES .......10

Name and other identifiers of the substance .............................................................................................. 10
Composition of the substance ........................................................................................................................... 10
Identity and composition of degradation products/metabolites relevant for the SVHC
assessment ....................................................................................................................................................... 10
Identity and composition of structurally related substances (used in a grouping or readacross approach)............................................................................................................................................. 10
Physicochemical properties ................................................................................................................................ 11
2. HARMONISED CLASSIFICATION AND LABELLING .....................................................................12
3. ENVIRONMENTAL FATE PROPERTIES ...............................................................................................12
Degradation .............................................................................................................................................................. 12
Abiotic degradation ........................................................................................................................................... 12
Biodegradation .................................................................................................................................................... 12
Environmental distribution ................................................................................................................................. 13
Adsorption/desorption ...................................................................................................................................... 13
Volatilisation ......................................................................................................................................................... 13
Distribution modelling ...................................................................................................................................... 13
Environmental occurrence data .................................................................................................................... 13
Human occurrence ............................................................................................................................................. 21
Summary on occurrence and environmental distribution .................................................................. 21
Bioaccumulation ..................................................................................................................................................... 21
Bioaccumulation in aquatic organisms (pelagic & sediment organisms) ...................................... 21
Bioaccumulation in terrestrial organisms (soil dwelling organisms, vertebrates) .................... 21
Summary ................................................................................................................................................................... 21
4. HUMAN HEALTH HAZARD ASSESSMENT ..........................................................................................21
5. ENVIRONMENTAL HAZARD ASSESSMENT .......................................................................................22
Aquatic compartment (including sediment) ................................................................................................. 22
Fish 22Aquatic invertebrates ....................................................................................................................................... 22
Algae and aquatic plants ................................................................................................................................. 23
Sediment organisms ......................................................................................................................................... 23
Other aquatic organisms ................................................................................................................................. 23
Terrestrial compartment...................................................................................................................................... 23
Toxicity to soil macro-organisms ................................................................................................................. 23
Toxicity to terrestrial plants ........................................................................................................................... 23
Toxicity to soil micro-organisms .................................................................................................................. 23
Toxicity to other terrestrial organisms....................................................................................................... 24
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ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC
Atmospheric compartment ................................................................................................................................. 24
Microbiological activity in sewage treatment systems ............................................................................. 24
Toxicity to birds ...................................................................................................................................................... 24
Mammalian wildlife ................................................................................................................................................ 24
6. ENDOCRINE DISRUPTION (ENVIRONMENT) ................................................................................25
General approach for the assessment of endocrine properties ............................................................ 25
Framework of the evaluation ......................................................................................................................... 25
Information sources and strategy for endocrine disruptor identification ..................................... 25
In vitro information indicative of endocrine activity ................................................................................. 26
In vivo mechanistic data with regard to an endocrine activity ............................................................ 34
In vivo adverse effect data with regard to an endocrine mode of action ......................................... 46
Conclusion regarding ED properties relevant for environment ............................................................ 62
Adverse effects relevant for ED identification ......................................................................................... 62
Endocrine activity ............................................................................................................................................... 65
Plausible link between adverse effects and endocrine activity ......................................................... 73
6. CONCLUSIONS ON THE SVHC PROPERTIES ...................................................................................77
CMR assessment..................................................................................................................................................... 77
PBT and vPvB assessment .................................................................................................................................. 77
Assessment under Article 57(f) ........................................................................................................................ 78
Summary of the data on the intrinsic/hazardous properties (providing scientific evidence of
probable serious effects to HH and/or ENV) ................................................................................ 78
Equivalent level of concern assessment .................................................................................................... 78
Conclusion on the Article 57(f) assessment ............................................................................................ 84
PART II .................................................................................................................................................................86
7. REGISTRATION AND C&L NOTIFICATION STATUS ....................................................................86
Registration status................................................................................................................................................. 86
CLP notification status .......................................................................................................................................... 86
8. TOTAL TONNAGE OF THE SUBSTANCE..............................................................................................87
9. INFORMATION ON USES OF THE SUBSTANCE ..............................................................................87
10. INFORMATION ON STRUCTURE OF THE SUPPLY CHAIN ......................................................88
11. ADDITIONAL INFORMATION .............................................................................................................88
REFERENCES FOR PART I .............................................................................................................................89

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ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHCTABLES
Table 1: Substance identity ...................................................................................... 10
Table 2: Overview of physicochemical properties 1 .................................................... 11
Table 3: Results for Surface water compartment ...................................................... 14
Table 4: Results for sediment compartment ............................................................. 15
Table 5: Results for waste water .............................................................................. 15
Table 6: Results for sewage sludge .......................................................................... 16
Table 7: Results for biota.......................................................................................... 16
Table 8: Descriptive data analysis for Naïades (suite) .............................................. 17
Table 9: Descriptive data analysis from Literature review ........................................ 18
Table 10: Summary table of in vitro mechanistic data (chronologic order) .............. 26
Table 11: Summary table of in vivo mechanistic data (chronologic order) ............... 34
Table 12: Summary table of in vivo adverse effect data (chronologic order) ............ 46
Table 13: Summary table of survival data - Japanese medaka (Oryzias latipes) ...... 60
Table 14: Summary table of survival data - Zebrafish (Danio rerio) ......................... 61
Table 15: Line of evidence in relation to TPhP sexual dysfunction in in-vivo studies 64
Table 16: Line of evidence in relation to TPhP EAS activity....................................... 67
Table 17: Dose-response and temporal concordance between the key events for malefish ........................................................................................................................... 74
Table 18: ELoC summary reporting ........................................................................... 82
Table 19 Registration status ..................................................................................... 86
Table 20: CLP notifications ....................................................................................... 86
Table 21: Tonnage status ......................................................................................... 87
Table 22: Uses .......................................................................................................... 87

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ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHCABBREVIATIONS:
adcy3 : adenylyl Cyclase 3
ALDH: aldehyde dehydrogenase
AO: adverse outcome
AR: androgen receptor
3β-HSD: 3β-hydroxysteroid dehydrogenase
17β-HSD: 17β-hydroxysteroid dehydrogenase
BBzP: benzyl butyl phthalate
BCF: bioconcentration factor
BOD: biochemical oxygen demandcAMP: cyclic adenosine monophosphate
CF: condition factor
CHO: chinese hamster ovary cells
C&L: classification and labelling
COS-7: simian kidney cells
CYP11A1: cholesterol side-chain cleavage
CYP11B2: aldosterone synthase
CYP17: 17α-hydroxylase
CYP19: cytochrome P450 aromatase
DEGs: differentially expressed genes
DEP: diethyl phthalate
DEPs: differentially expressed proteins
DHEA: dehydroepiandrosterone
DiBP: diisobutyl phthalate
Dio1: thyroxine deiodinase, type I
Dio2: thyroxine deiodinase, type II
dpf/h: days post fertilisation/hatch
DT50: degradation half-life time
dw: dry weightE2: 17β-Estradiol
E2-F: fluorescent derivative of 17β-Estradiol
EAS: Estrogen/Androgen/ Steroidogenesis (modalities)
EATS: Estrogen/Androgen/Thyroidal/Steroidogenesis (modalities)
EC ED EAG: Expert Advisory Group of the European Commission on Endocrine Disruptor
EC10: 10% effective concentration
EC20: 20% effective concentration
EC50: half maximal effective concentration
ECHA: European Chemicals Agency
ED: endocrine disruptor
EDC: endocrine disrupting chemical
EDC-WG: ANSES’ Thematic Working group on Endocrine Disruptors
EFSA: European Food Safety Authority
EGFR: epidermal growth factor receptor
ELoC: equivalent level of concern
ER: estrogen receptor
ERE: estrogen response element
FRTL: rat thyroid follicular cell strain
FSDT: fish sexual development test
G15: GPR30 inhibitor
GC/MS: gas chromatography/ mass spectrometry
GH3: rat pituitary-derived cell line that expresses the Pit-1 transcription factor.
GPER: G protein-coupled estrogen receptor
GR: glucocorticoid receptor
GS: Gosner stage
GSI: gonadosomatic index (gonadal weight/body weight x 100)
H295R: Human adrenocarcinoma cell line
HEK293: immortalised human embryonic kidney cells
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ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC
HepG2: Human liver cancer cell line
HG5LN: HeLa cells with the (GAL4RE) 5-betaGlobin-Luciferase-SV40-Neomycin plasmid
HMGR: 3-Hydroxy-3-methylglutaryl-coenzyme A reductase
hpf: hours post-fertilisation
HPLC: high performance liquid chromatography
HPSE: Heparanase
HSD3β2: 3β-hydroxysteroid dehydrogenase type 2
HSI: hepatosomatic index
IC50: concentration required to inhibit the cell viability by 50%
JRC: Joint Research Centre
KEs: key events
Koc: organic carbon-water partition coefficient
Kow: octanol/water partition coefficient
kPa: kilopascal
11-KT: 11-ketotestosterone
LC: liquid chromatography
LC10: concentration inducing 10% lethality
LC50: concentration inducing 50% lethality
Lhcgr: luteinising hormone/choriogonadotropin receptor
LOD: limit of detection
LOEC: lowest observed effect concentration
LOQ: limit of quantification
MA-10: mouse Leydig cell line tumor
MAPK: mitogen-activated protein kinase
MBzP: monobenzyl phthalate
MCF-7: breast cancer cell line (Michigan Cancer Foundation-7)
MEP: monoethyl phthalate
MIE: molecular initiating event
MoA: mode of action
MR: mineralocorticoid receptor
MS: mass spectrometry
MSCA: member state competent authority
MTC: maximum tolerated concentration
MVLN ERα-positive and hormone-responsive human breast carcinoma cell line derived from theMCF-7 cell line, stably transfected with an ER-controlled luciferase reporter gene construct
nd: not detected
nER: nuclear estrogen receptor
NIS: sodium/iodide symporter
Nkx2.1: NK2 homeobox 1 also known as thyroid transcription factor 1 (TTF-1).
NO(A)EL: no observed (adverse) effect level
NOEC: no observed effect concentration
NR: not reported.
OEL: occupational exposure limit
Pax8: paired box gene 8
PI3K-Akt: phosphoinositide 3-kinase/protein kinase B
POMC: proopiomelanocortin receptor
PPARγ: peroxisome proliferator-activated receptor-γ
PXR: pregnane X receptor
QSAR: Quantitative Structure-Activity Relationship
RT-PCR: real-time polymerase chain reaction
TWA: time weighted average
RA: retinoic acid
RAR: retinoic acid receptor
RBA: relative binding affinity
RDH: retinal dehydrogenase
REC/RIC20: response equal to 20 % agonist/inhibitory activity
RGA: reporter gene assay
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ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC
RPE: relative proliferative effectSKBR3: human breast cancer cell line isolated by the Memorial Sloan–Kettering Cancer
Center in 1970
SOD: superoxide dismutase
SPM: suspended particulate matter
SRC: steroid receptor co-activators
SSC: secondary sex characteristics
StAR: steroidogenic acute regulatory protein
SULT: sulfotransferase
SVHC: substance of very high concern
T3 : 3,5,3-triiodo-L-thyronine
T4: L-thyroxine
T: testosterone
Tg: thyroglobulin
TPhP: triphenyl phosphate
TPO: thyroperoxidase
TR: thyroid hormone receptor
TSH: thyroid-stimulating hormone (thyrotropin)
TSHR: thyroid-stimulating hormone receptor (thyrotropin receptor)
TSPO: translocator protein
TTR: transthyretin receptor
UHPLC–MS/MS: ultra-high performance liquid chromatography tandem mass spectrometry
US: United States of America
US EPA: Environmental Protection Agency of the United States of America
VTG: vitellogenin (protein)
vtg: vitellogenin (gene)
WHO/IPCS: International Program on Chemical Safety of the World Health Organisation
ww: wet weight

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ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHCPROPOSAL FOR IDENTIFICATION OF A SUBSTANCE
OF VERY HIGH CONCERN ON THE BASIS OF THE
CRITERIA SET OUT IN REACH ARTICLE 57
Substance name: Triphenyl phosphate (TPhP)EC number: 204-112-2
CAS number: 115-86-6•

It is proposed to identify the substance as a substance of equivalent level of concern tothose of other substances listed in points (a) to (e) of Article 57 of Regulation (EC) No
1907/2006 (REACH) according to Article 57(f) of REACH Regulation.

Summary of how the substance meets the criteria set out in Article 57 of the REACH
Regulation
TPhP is proposed to be identified as a substance of very high concern in accordance with Article
57(f) of Regulation (EC) 1907/2006 (REACH) because it is a substance with endocrine disrupting
properties for which there is scientific evidence of probable serious effects to the environment,
which give rise to an equivalent level of concern to those of other substances listed in points (a)to (e) of Article 57 REACH.
The available in vitro information demonstrates the capacity of TPhP to competitively bind andactivate nuclear estrogen receptors ERα and ERβ of several vertebrate species including rat,mouse, fish, chicken, frog and turtle as evidenced by ER transactivation in reporter cell lines. In
addition, TPhP can induce ER-regulated gene expression, and related physiological cell responses(e.g., increased cell proliferation). Two recent studies show that TPhP can also bind and activate
GPER. The available H295R assays on human adrenal carcinoma cells show that TPhP affects
steroidogenesis by increasing estrogen levels (17ß-estradiol) and by increasing expression ofgenes involved in this pathway like CYP19 and 3β-HSD2. In vivo fish studies indicate that CYP19A
is significantly upregulated by exposure to TPhP. Significant alteration of plasmaticconcentrations of E2 and E2/T ratio (more specific observation in females) and E2/11-KT ratio(more specific observation in males) can result from this modification in the steroidogenesis
pathway. The degree of perturbation of circulating steroid concentrations depends on the fish
developmental stage, species and tested concentrations. The observations of VTG
concentrations, that are consistent with perturbation of E2 concentrations, suggest an EAS
activity of TPhP in female and male zebrafish, with altered plasmatic concentration of VTG.
Therefore, TPhP exerts an effect on the endocrine balance in fish. It has EAS activity as clearlyshown both in vitro and in vivo.
In vivo studies on fish reproduction show an alteration of gametogenesis in both sexes with adisruption of testis and ovary maturation followed by a decrease in reproductive success in termsof fecundity (decrease in egg production, spawning events fertility (impaired hatchability) andfertilisation rate). Therefore, TPhP shows adverse effects on fertility and reproduction in fish,observed in several studies at levels without concurrent systemic effects.
The consistency between the observed adverse effects and EAS activity provides evidence that
EAS modalities are plausibly biologically linked to the adverse effects. Depending on thedevelopmental stage, species and concentration, antagonist and agonist effects are observed inorganisms, leading in vivo to perturbations of circulating steroid concentrations in most of theanalysed studies.

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ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC
The effect observed on reproduction in fish (fecundity and fertility) can affect population stability
and is considered as an adverse effect relevant at population level.
Based on all available scientific evidence, it can be concluded that TPhP fulfils the WHO/IPCS
definition of an endocrine disruptor.
The very high concern raised by this property is substantiated by the severity and irreversibilityof the effects on organisms and populations that may have long term consequences, the large
variety of species that may be adversely affected and the difficulties to quantify a safe level of
exposure with regard to the endocrine mediated effects.
In conclusion, there is scientific evidence that TPhP causes probable serious effects to theenvironment due to its endocrine disrupting properties, which give rise to an equivalent level ofconcern to those of other substances listed in points (a) to (e) of Article 57 of the REACH
Regulation.
Registration dossiers submitted for the substance: Yes

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ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHCPART I
Justification
1. Identity of the substance and physical and chemical
properties
Name and other identifiers of the substance
Table 1: Substance identity
EC number:

204-112-2

EC name:triphenyl phosphate

CAS number (in the EC inventory):

115-86-6

CAS number:

115-86-6

IUPAC name:triphenyl phosphateIndex number in Annex VI of the CLP
Regulation

None

Molecular formula:

C18H15O4P

Molecular weight range:

326.28

Synonyms:

TPhP

Structural formula:Composition of the substance
Name: Triphenyl phosphate
Description: Colourless, odourless solid.
Substance type: mono-constituent

Identity and composition of degradation products/metabolites relevant
for the SVHC assessment
Not relevant for the SVHC assessment of the substance.

Identity and composition of structurally related substances (used in agrouping or read-across approach)
Not applicable.
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ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC

Physicochemical properties
Table 2: Overview of physicochemical properties
Property

Description of key
information

Value [Unit]

Physical state at
20°C and 101.3
kPa

Visual inspection

Colourless, odourless solid.

Melting/freezing
point

Non-guideline
publication

49.5 - 50 °C. Melting pointvalues within the range 49 50.5 °C are reported in avariety of secondary
sources.

Boiling point

Extrapolated
according to theClausius-Clapeyron
equation using
experimentallyderived parameters non-guideline
publication.

414 °C at 101.3 kPa. One
publication reported
decomposition at or near
the boiling point.

Vapour pressure

Non-guideline
publication using an
isoteniscope under anitrogen atmosphere.
Results extrapolatedusing the ClausiusClapeyron equation.

0.000853 Pa at 25°C

Density

Non-guideline
publication.

The relative density at 50°C
is given as 1.21g/cm3.

Water solubility

Non-guideline
publication.

1.9 mg.l-1 at 20°C. Other
supporting
publications/reference
sources give similar values.
No modern guideline study.

Partitioncoefficient noctanol/water(log value)

Non-guideline but
similar to shake-flask.

Log Pow 4.63. Values
between 4.5 and 4.7 arereported in various
publications

Granulometry

OECD Guideline 110(Particle Size
Distribution / Fibre
Length and Diameter
Distributions).

All particles with a mean
diameter < 100 μm have amass fraction of 0.41 %.

Reference/sourceof information
Data quoted from the
FR conclusion
document1 publishedin:https://echa.europa.e
u/documents/10162/9
16779d9-ec10-07faf178-9562bdd7dedc

0.023 % / 0.019 %(spherical/ cubical) particlesof this mass fraction have amean diameter < 4 μm.

1

Physicochemical properties quoted in the Conclusion document were based on the analysis ofphysicochemical properties extracted from the registration dossier of the lead registrant as disseminatedin
ECHACHEM(https://chem.echa.europa.eu/100.003.739/dossier-view/9b2f054a-c82b-4cc1-9393415a4f7781c1/4c37f1df-d04d-4911-8afc-4a9bf3b52bf0_4c37f1df-d04d-4911-8afc4a9bf3b52bf0?searchText=204-112-2).

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ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC

2. Harmonised classification and labelling
No current harmonised classification for TPhP.

3. Environmental fate properties
The environmental fate data presented in this section are extracted from the registration dossierof the lead registrant as disseminated in ECHACHEM1 and are provided as contextual information
for the SVHC assessment of the substance that focus on endocrine disruptor (ED) properties forthe environment. These data have not been further evaluated.

Degradation
Abiotic degradation
Hydrolysis
The main study gave half-lives of 3 days at pH 9, 19 days at pH 7 and >28 days at pH 5 at 25°C
(Mayer et al., 1981). One study indicates that 100 % of the TPhP is hydrolysed after 10 minutesat pH 13 (Ishikawa et al., 1985). Another study indicates that at pH 4 the rate of disappearance
is too slow to measure (Howard et al., 1979). All studies show that under alkaline conditions
TPhP is rapidly degraded, while under acidic conditions TPhP is relatively stable.

Phototransformation in air
From AOPWIN v1.92, a calculation based on a 12 hour-day and a concentration of
1.5x10E6 OH/cm3 gave a half-life of TPhP of 11.85 hours.

Phototransformation in water
Two studies (Ishikawa et al., 1992; Wan and Wong, 1994) are cited for phototransformation inwater, and the registrant concluded that TPhP was rapidly degraded when irradiated at 254 nm.
The key test was performed at pH 3 and pH 10. However, since the stratospheric ozone layer
prevents ultraviolet light of less than 290 nm from reaching the earth's surface, only light atwavelengths between 290 and 750 nm can result in photochemical transformations in theenvironment.

Phototransformation in soil
No relevant information available.

Biodegradation
Biodegradation: screening tests
Eight biodegradation screening studies are summarised in the registration dossier. These include
tests of both ready and inherent biodegradation, as well as several river and pond sedimentassays. The key study is CITI (1992), which is an OECD 301C test where biodegradation between
83-94% (based on BOD after 28 days was observed).

1

https://chem.echa.europa.eu/100.003.739/dossier-view/9b2f054a-c82b-4cc1-9393-415a4f7781c1/4c37f1df-d04d4911-8afc-4a9bf3b52bf0_4c37f1df-d04d-4911-8afc-4a9bf3b52bf0?searchText=204-112-2

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ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC

Biodegradation in water and sediment
An OECD 303A (sewage treatment plant simulation study) is included assessing aerobic
degradation (Unpublished study report, 1982). The registrant concludes that TPhP is readily
degraded in water/sediment systems.

Biodegradation in sediment
No data available

Biodegradation in soil
Two simulation tests for biodegradation in soil (aerobic and anaerobic conditions) aresummarised in the registration dossier (Anderson et al., 1993). They calculated a half-life (DT50)
of 37 days in aerobic conditions and 21 days in anaerobic conditions.

Environmental distribution
Adsorption/desorption
Four studies in the registration dossier provide information on adsorption/desorption (Anderson
et al., 1993; Mayer et al., 1981; Boethling & Cooper, 1985; Huckins et al., 1991). The registrant
notes that the range of values (Koc between 2514 and 5500) indicates that the substance will
be relatively immobile in soil. The key data is Anderson et al. (1993) using three soils, whichgave measured Koc values between 2414 and 3561.

Volatilisation
The Henry’s law constant has been calculated by QSAR using HENRYWIN v3.2. The estimatedHenry’s law constant is 0.004 Pa.m3/mol.

Distribution modelling
The registrant includes Mackay Level 1 modelling (Unpublished study report, 2002), which
provides the following distribution:
Air:
Water:
Soil:
Sediment:
Suspended sediment:
Biota:
Aerosol:

0.7%
14.3%
43.9%
41%
0.07%
0.03%
0%

The following inputs were used: temperature = 25 °C, vapour pressure = 8.35 x 10E-4 Pa, watersolubility = 1.90 g/m3, log Kow = 4.59.

Environmental occurrence data
This section integrates data from i) the Norman EMPODAT database, ii) the French Naïades
database and, iii) a review of the scientific literature.

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ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC

Norman EMPODAT database
Norman EMPODAT is a database of geo-referenced monitoring and bio-monitoring data in Europe
on emerging substances in the following matrices: water, sediment, biota, SPM, soil, sewage
sludge and air. This database cannot be used as representative of contaminated areas associatedto industrial activities, but rather as an initial overview of the global state of various
environmental media.
Surface water - results
24 580 samples, mainly from three countries (France, Germany and Netherlands) are provided
in this database. It should be noted that the variability of the detection and of the quantification
limits restrains the data exploitation. 8% of data was reported with no information about thelimit of quantification, 85% of values with LOQ > = 0.01 µg.l-1 and 24% of values with LOQ >=0.1 µg.l-1.
Table 3: Results for Surface water compartment
Descriptive data analysis for surface water – (2002 – 2020)
Total individual values > LOQ: 1835 (7.47% of total analysis (24580))
Parameter

Concentration Value (µg.l-1)

90th Percentile

5.42E-02

Mean

2.79E-02

Median1.50E-02

Max

1.74

Surface water data distribution per country
45.00%
40.00%
35.00%
30.00%
25.00%
20.00%
15.00%
10.00%
5.00%
0.00%

Individual ValueLess than LoD

Less than LoQ

Figure 1: Percentage of surface water data (Total number of analysis) distribution per country

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ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC
Sediment - results
Forty samples from eight countries (Moldova, Ukraine, Bulgaria, Georgia, Germania, Hungary,
Romania, and Montenegro) are provided in this database for TPhP with 4 values above the LOD(1.5 µg/kg dw). The other 36 samples had a LOD of 5 µg/kg dw.
Table 4: Results for sediment compartment
Descriptive data analysis for sediment – values above the LOD

COUNTRY

Ecosystem/ Matrix:
sediment

Germany
Hungary
Bulgaria

Concentrationvalue
6

Sediments River water

Ukraine

9
9

Unit

Month

Year

River
basin
name

µg/kg
dry
weight7

2019

DanubeLimit of
Detection(LOD):[µg/kgdw]

Analyticalmethod1.5

HPLC-MSor MS/MS

5

Waste water – results
The data for waste water covers the year 2007 and 3 consecutive years (2017-2018-2019). The
range of the detection limits and the quantification limits in µg.l -1 are [8E-05; 5E-02] and [2.5E04; 6E-02] respectively.
Table 5: Results for waste water
Descriptive data analysis for waste water
Total individual value (> LOQ): 29 (28.2% of total analysis)
Total number of analysis: 103
Parameter

Concentration Value (µg.l-1)

90th Percentile

7.86E-02

Mean

3.52E-02

Median1.77E-02

Max

1.96E-01

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ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC

Waste water data distribution per country
45.00%
40.00%
35.00%
30.00%
25.00%
20.00%
15.00%
10.00%
5.00%
0.00%
Austria Bulgaria Croatia

Czech Germany Hungary Romania Serbia
Republic
Individual ValueLess than LoD

Slovakia Slovenia Ukraine United
KingdomLess than LoQ

Figure 2: Percentage of waste water data (total number of analysis) distribution per country

Sewage sludge - results
Eight samples from Sweden only are provided in this database for TPhP. No information about
the limit of quantification or detection is available.
Table 6: Results for sewage sludge
Descriptive data analysis for sewage sludge
Type

Value

Unit

Month

Year

AnalyticalmethodSewage sludge - Mixed

54µg/kg dry weight

11

2007

GC-MS or MS/MS

Sewage sludge - Mixed

265µg/kg dry weight

10

2007

GC-MS or MS/MS

Sewage sludge - Mixed

54µg/kg dry weight

10

2007

GC-MS or MS/MS

Sewage sludge - Mixed

136µg/kg dry weight

10

2007

GC-MS or MS/MS

Sewage sludge - Municipal

107µg/kg dry weight

10

2007

GC-MS or MS/MS

Sewage sludge - Industrial

32µg/kg dry weight

2

2008

GC-MS or MS/MS

Sewage sludge - Mixed

104µg/kg dry weight

10

2007

GC-MS or MS/MS

Sewage sludge - Municipal

1306µg/kg dry weight

10

2007

GC-MS or MS/MSCountry

SwedenBiota – results
Among the 205 samples available in this database for TPhP, 2 samples on marine mammals andraptors are above the LOD (range value [0.00125; 5] µg.kg ww-1).
Table 7: Results for biota
Descriptive data analysis for biota (205 samples with 2 samples > LOD)CountryBelgium
Belgium

Sample matrix
Biota - Territorial (marine)
water
Biota - Terrestrial

Valu
e

Unit

Year

0.99

µg/kg wet weight

2021

0.81

µg/kg body weight

2021

Species
group
Marinemammals
Raptors

SpeciesnamePhocoena
phocoena
Buteo

16 (95)

ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHCNaïades databaseNaïades is a database collecting French data only. It is an observatory on the quality of river andwater bodies. 4705 samples are included in the database for TPhP between 2020 and 2022,
including 4647 values below the limit of quantification (LOQ) and 58 values above the LOQ(range: 0.02-0.1 µg.l-1). These values (> LOQ) were measured in a single French region and bya single analysis laboratory.
Table 8: Descriptive data analysis for Naïades (suite)
Descriptive data analysis for value > LOQ (58 values)
Parameter

Raw water concentration (µg.l-1)

90th Percentile

0.12

Mean

0.06

Median

0.046

Max

0.22

Scientific literature related to occurrence data
A scientific literature review was conducted up to May 2023. The systematic literature search
was performed in Scopus database. A single concept strategy search was applied to retrieve all
relevant information on TPhP by using its Chemical Abstracts Service Registry Number(CAS No 115-86-6), scientific chemical names, and common names, as recommended in the
EDC guidance (ECHA/EFSA, 2018). 812 entries were recorded from 23 studies that included
monitoring data in an environmental media (water, sediment, biota) in Europe.

17 (95)

Table 9: Descriptive data analysis from Literature review
Descriptive data analysis from Literature review
CountryType ofenvironmental media

Type of value

Value

Mean (min-max)

1.7(1.6-2.4)
2.8(1.6-7.2)
12.5(01.6-35)

Unit

LOD

AnalyticalmethodYearng.l-1

1.6 ng.l-1

GC-MS

2012 (11 samples)ng.l-1

1.6 ng.l-1

GC-MS

2012 (8 samples)ng.l-1

1.6 ng.l-1

GC-MS

2012 (13 samples)ng.l-137 ng.l-1

GC-MS-MS2013(25 samples)

Gustavsson etal., 2018

ng.l-1NR

2019-2020(12 samples)

Pantelaki etal., 2021

ng.l-1NR

2019-2020(12 samples)

Pantelaki etal., 2021

ng.l-1NR

2019-2020(18 samples)

Pantelaki etal., 2021

ng.l-1NR

2019-2020(18 samples)

Pantelaki etal., 2021

ng.l-1NR

2019-2020(12 samples)

Pantelaki etal., 2021

ng.l-1NR

2019-2020(12 samples)

Pantelaki etal., 2021

2019-2021(12 samples)
2019-2021(12 samples)
2019-2021(12 samples)

Onoja et al.,2023
Onoja et al.,2023
Onoja et al.,
2023

REF

WATER
Spain

Nalon river

Spain

Arga river

Spain

Besos river

Sweden21 rivers

GreeceRiver water
Dissolved fraction

GreeceRiver water
Particulate fraction

GreeceCoastal water
Dissolved fraction

GreeceCoastal water
Particulate fraction

GreeceStreams
Dissolved fraction

GreeceStreams
Particulate fraction

Mean (min-max)
Mean (min-max
Mean

< LOD

Mean (min-max)
Detection
frequencies
Mean (min-max)
Detection
frequencies
Mean (min-max)
Detection
frequencies
Mean (min-max)
Detection
frequencies
Mean (min-max)
Detection
frequencies
Mean (min-max)
Detection
frequencies

137(40-258)100%
100(46-219)100%
135(41-260)100%
47(34-81)100%
279(45-1142)100%
281(38-980)100%

Mean (min-max)

4(0.1-26)1(0.3-8)1(0.4-2)

GC-MS-MS
GC-MS-MS
GC-MS-MS
GC-MS-MS
GC-MS-MS
GC-MS-MS

Cristale et al.,2013
Cristale et al.,2013
Cristale et al.,2013

SEDIMENT
United Kingdom

Worcester-Birmingham
Canal

United Kingdom

River Severn

United Kingdom

River Sowe

Mean (min-max)
Mean (min-max)

ng/g dryweightng/g dryweightng/g dryweightNR

GC-MSDNR

GC-MSDNR

GC-MSDANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC
United Kingdom

River Tame

Spain

Nalon

Spain

Arga

Spain

Besos

Mean (min-max)
Mean (min-max)
Mean (min-max)
Mean (min-max)

2(0.3-9)15(15-15)22.17(15-44)37.71 (1963)

ng/g dryweightµg/kg dryweightµg/kg dryweightµg/kg dryweightNR

GC-MSD2019-2021(12 samples)

15 µg/kg dw

GC-MS2012 (8 samples)

15 µg/kg dw

GC-MS2012 (6 samples)

15 µg/kg dw

GC-MS2012 (7 samples)

0.08 ng/g
dw

Italy

Adige River

Max

9.69

ng/g dryweightGreeceEvrotas River

Max

0.67

ng/g dryweight0.08 ng/gdw

Slovenia

Sava River

Max

< LOD

ng/g dryweight0.08 ng/g
dw

FranceGulf of Lion

Mean
(Min-Max)

1.57(0-5.17)

ng/g dryweightNR

Mussels (Mytilus spp)
and oysters(Crassostrea gigas)

Mean or Median(Min-Max)

1.12(0.17-8.01)

ng/g dryweight53 pg/g dw

Pressurised liquid
extractionTurbulent Flow
ChromatographyLC-MS/MS
Pressurised liquid
extractionTurbulent Flow
ChromatographyLC-MS/MS
Pressurised liquid
extractionTurbulent Flow
ChromatographyLC-MS/MS

Onoja et al.,2023
Cristale et al.,2013
Cristale et al.,2013
Cristale et al.,20132015 (20 samples)

Giulivo et al.,2017

2014-2015 (12samples)Giulivo et al.,2017

2014-2015 (20samples)Giulivo et al.,
2017

GC-MS

2018
(12 samples)

Alkan et al.,2021

LC-ESI-MS/MS

2014-2021

Aminot et al.,2023

2015 (13 samples)

Giulivo et al.,20172015 (4 samples)

Giulivo et al.,20172015 (10 samples)

Giulivo et al.,
2017

BIOTA
France

Italy

Adige River(Fish)

Max

30.3

ng/g lipid
weight1.30 ng/g lw

GreeceEvrotas River(Fish)Max< LOD

ng/g lipid
weight1.30 ng/g lw

Slovenia

Sava river(Fish)Max< LOD

ng/g lipid
weight1.30 ng/g lw

Pressurised liquid
extractionTurbulent Flow
ChromatographyLC-MS/MS
Pressurised liquid
extractionTurbulent Flow
ChromatographyLC-MS/MS
Pressurised liquid
extractionTurbulent Flow
ChromatographyLC-MS/MS

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ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC
Mean

59.2

ng/g lipid
weightNR

Ultra-sonication
extraction-LCMS/MS

2015(20 whales)

Garcia-Garinet al. 2020

Mean
(Min-Max)

2.42(ND-9.39)

ng/g wetweight0.12 ng/g
ww (LOQ)

Ultra-sonication
extraction-TFCHPLC-MS/MS

2019

Sala et al.,
2022

Mean
(Min-Max)

0.66(ND-2.28)

ng/g wetweight0.12 ng/g
ww (LOQ)

2019

Sala et al.,
2022

Mean
(Min-Max)

ND

ng/g wetweight0.12 ng/g
ww (LOQ)

2019

Sala et al.,2022

TurtleMean(Min-Max)

0.15(ND-1.08)

ng/g wetweight2014-2017

Sala et al.,2021

Mediterranean sea(Balearic Island)

TurtleMean(Min-Max)

0.31(ND-1.34)

ng/g wetweight2014-2017

Sala et al.,2021

Mediterranean sea(Balearic Island)

Prey of turtle

Mean(Min-Max)

1.25

ng/g wetweight2014-2017

Sala et al.,2021

Denmark
Western
Mediterranean area
Western
Mediterranean area
Western
Mediterranean area
Mediterranean sea(Catalan coast)

Western Iceland(Denmark Strait)
Fin Whale
Western Mediterranean
Sea
Sardina Pilchardus
Western Mediterranean
Sea
Engraulis Encrasicolus
Western Mediterranean
Sea
Merluccius Merluccius

0.12 ng/g
ww (LOQ)
0.12 ng/g
ww (LOQ)
0.12 ng/g
ww (LOQ)

Ultra-sonication
extraction-TFCHPLC-MS/MS
Ultra-sonication
extraction-TFCHPLC-MS/MS
Ultra-sonication
extraction-TFCHPLC-MS/MS
Ultra-sonication
extraction-TFCHPLC-MS/MS
Ultra-sonication
extraction-TFCHPLC-MS/MS

NR: not reported.

20 (95)

Human occurrence
In addition to indirect exposure via the environment, humans are exposed via several consumer
articles or in the workplace (widespread uses), in formulation or repacking, on industrial sites
and in manufacturing. TPhP is also present as an impurity in many other organophosphate flame
retardants or as a constituent of this family of compounds.
Human biomonitoring data demonstrate also the presence of TPhP or its metabolites in humanmilk (Sundkvist et al., 2010; Kim et al., 2014; Zheng et al., 2021), human placenta (Ding et al.,2016; Zhao et al., 2017), blood serum (Ya et al., 2019; Zhao et al., 2016) and urine (Carignan
et al., 2016; Li N. et al., 2019a). TPhP has also frequently been detected in indoor house dust
samples in Europe (Belgium and Sweden) and outside Europe (Japan, Philippines and USA) with
detection frequencies up to 98% (Kanazawa et al., 2010; Kim et al., 2013; Marklund et al.,
2003; Stapleton et al., 2009; Van den Eede et al., 2011). It can be concluded that TPhP reaches
diverse environmental compartments and biota of remote areas.

Summary on occurrence and environmental distribution
Several literature studies demonstrate that TPhP can be found in Europe and that it is ubiquitousin several environmental compartments (water, sediment, sewage sludge, indoor dust/air).
Therefore, many environmental species and humans are exposed more or less continuously to
TPhP and exposure cannot be avoided.

Bioaccumulation
Bioaccumulation in aquatic organisms (pelagic & sediment organisms)
The registration dossier of the Lead Registrant cites 9 fish bioaccumulation studies (Muir et al.,
1980; Mayer et al., 1981; Sasaki et al., 1981; Muir et al., 1982; Sasaki et al., 1982; Muir et al.,
1983; Boethling & Cooper, 1985; Kuehl and Haebler , 1995; Lo et al. 2000), although only threewere assessed to be valid of which two are conventional tests (the third was blubber samples
from dead Common bottlenose dolphins). These two conventional tests provide bioconcentration
factor (BCF values of 110 and 144.

Bioaccumulation
vertebrates)interrestrialorganisms(soil

dwelling

organisms,

No relevant information available.

Summary
Considering data coming from the registration dossier of the lead registrant, the substance israpidly degraded in water under alkaline conditions, TPhP photodegrades rapidly in theatmosphere (DT50= 11.85 h) and TPhP would be readily biodegraded in water/sediment
systems. The data provided in the registration dossier of the Lead registrant would indicate that
TPhP is relatively immobile in soil.

4. Human health hazard assessment
Human health data for TPhP were not reviewed and thus not included in this document.

ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC

5. Environmental hazard assessment
Please note that the environmental toxicity data presented in this section are extracted from theregistration dossier of the Lead Registrant as disseminated in ECHACHEM 2 and are provided ascontextual information for the SVHC assessment of the substance that focuses on ED properties
for the environment. These data have not been further evaluated. However, the data used as abasis for the environmental assessment of the potential endocrine disrupting properties of TPhP
have been evaluated and are described in section 5.

Aquatic compartment (including sediment)
Fish
Short-term toxicity to fish
Twelve studies are reported in the registration dossier. All of these studies are old (generally
performed more than thirty years ago) and mostly used static exposure conditions. Three include
chemical analysis, although the results are only reported for one.
The registrant judges the majority of the tests to be Klimisch reliability 2 with two consideredunreliable (Klimisch 3 and 4). The reliable studies report a range of LC 50 values between >0.32
and 1.26 mg.l-1, with the majority below 1 mg.l-1 (Mayer et al., 1981; Palawski et al., 1983;
Huckins et al., 1991; Sasaki et al., 1981). The lowest, reliable value is a 96-h LC50 of 0.36 mg.l1
for Rainbow Trout Oncorhynchus mykiss (Palawski et al., 1983). However, the key study
selected by the registrant is Mayer et al. (1981), which, reported a 96-h LC50 of 0.40 mg.l-1 for
O. mykiss based on nominal concentrations.

Long-term toxicity to fish
There are five long-term studies in the registration dossier, all of which are judged to be Klimischreliability 2 by the registrant. However, two of these are 96 h tests which should not be
considered as part of the chronic dataset. Of the remaining three tests, the key study for longterm toxicity to fish (Sitthichaikasem 1978) started with 10-days old sac frys and lasted 30 days,
and is considered part of an Early Life Stage Test. No NOEC was obtained with the TPhP
concentrations tested.

Aquatic invertebrates
Short-term toxicity to aquatic invertebrates
A test on the acute toxicity of TPhP to Daphnia magna was conducted according to the US
guideline EPA-660/3-75-009. Static exposure for 96 h resulted in a LC 50 value of 1.0 mg.l-1(nominal concentration). Lower effective concentrations were found for another crustacean,
Mysidopsis bahia, in the same test on acute toxicity. Static exposure for 96 h resulted in a LC 50
value of 0.18 mg.l-1 (nominal concentration) (Mayer et al., 1981).
Further invertebrate toxicity studies investigated the acute effects of TPhP on scud (Gammarus
pseudolimnaeus) and midge larvae (Chironomus riparius) (Huckins et al., 1991). Static exposurefor 96 h resulted in a LC50 value of 0.25 mg.l-1 and 0.36 mg.l-1 respectively (nominal
concentrations). Further, Lo et al., 2000 investigated the toxicity of TPhP to golden apple snail

2

https://chem.echa.europa.eu/100.003.739/dossier-view/9b2f054a-c82b-4cc1-9393-415a4f7781c1/4c37f1df-d04d4911-8afc-4a9bf3b52bf0_4c37f1df-d04d-4911-8afc-4a9bf3b52bf0?searchText=204-112-2

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ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC
(Pomacea canaliculata) and reported a relatively lower toxicity than identified for crustaceans inother studies (72 hr, LC50 38.2 mg.l-1).

Long-term toxicity to aquatic invertebrates
The chronic toxicity of TPhP to aquatic invertebrates (Daphnia magna) was tested according toOECD Guideline 211 (Daphnia magna Reproduction test). After 21 days a NOEC of 0.254 mg.l-1(mean measured concentration) was obtained based on reproduction (Unpublished study report,
2000).

Algae and aquatic plants
The registrant reports nine tests. Eight of these tests appear to be the same as those reported
in Environment Agency (2009). The registrant judges all of the tests to be Klimisch reliability 2,
which is consistent with Environment Agency (2009).
The registrant considers the test (three species and three different media) conducted byMillington et al. (1988) according to an OECD guideline 201 to be the key study. The authors of
the test only reported LOEC values. The most critical values were obtained for Desmodesmus
subspicatus and Pseudokirchneriella subcapitata. The data reports LOECs between 0.5 and
5.0 mg.l-1 depending on the growth medium. Mayer et al. (1981) determined the toxicity of TPhP
to Desmodesmus subspicatus and a 96h-EC50 was determined to be 2 mg.l-1.

Sediment organisms
No relevant information available.

Other aquatic organisms
No relevant information available.

Terrestrial compartment
Toxicity to soil macro-organisms
No relevant information available.

Toxicity to terrestrial plants
No relevant information available.

Toxicity to soil micro-organisms
No relevant information available.

23 (95)

ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC

Toxicity to other terrestrial organisms
No relevant information available.

Atmospheric compartment
No relevant information available.

Microbiological activity in sewage treatment systems
The registrant used an activated sludge assay as key study, according to OECD guideline 301C
(CITI, 1992). Activated sludge (30 mg.l-1 as concentration of suspended solid) in a volume of
300 mL was exposed to TPhP (100 mg.l-1) for 28 days at 25°C. Degradation was determined by
BOD. Under the used conditions, TPhP did not adversely affect the microorganisms present, since
TPhP biodegraded to the extent of 83 - 94 %. The key values should be understood as > 100
mg.l-1 as no effect was observed up to the test concentration of 100 mg.l-1.

Toxicity to birds
No relevant information available.

Mammalian wildlife
No relevant information available.

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ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC

6. Endocrine disruption (Environment)
General approach for the assessment of endocrine properties
Framework of the evaluation
To evaluate whether or not TPhP fulfils the WHO/IPCS definition (WHO/IPCS, 2002) of an
endocrine disruptor as interpreted by the EC ED EAG (JRC, 2013), both in vitro and in vivo data
were taken into account, in order to demonstrate:• Adverse effects• Endocrine mode of action• Biological plausible link between adverse effects and endocrine mode of action•
As highlighted in EDC guidance developed by ECHA and the European Food Safety Authority(EFSA) to identify EDC under the plant protection products and the biocidal product regulations
published in 2018 (ECHA/EFSA, 2018), the ‘endocrine mode of action’ in the second bullet pointshould be interpreted as ‘endocrine activity’, i.e., the substance has the potential to alter thefunction(s) of the endocrine system. Consequently, the third bullet point should be interpreted
as biological plausible link between adverse effects and endocrine activity.
Specificity, i.e., that adverse effects were not non-specific secondary consequences of general
toxic effects, and population relevance were also considered in the assessment.
The structure and the assessment of data are mainly based on the OECD Revised GuidanceDocument 150 on standardised test guidelines for evaluating chemicals for endocrine disruption(OECD, 2018).
Two different aspects are assessed separately:• Evidence for endocrine activity• Effects on apical endpoints that provide evidence that a substance exerts adverse
effects owing to its endocrine activity.

Information sources and strategy for endocrine disruptor identification
Literature review
A literature review was performed following the principles displayed in the EDC guidance(ECHA/EFSA, 2018). The EDC guidance provides a tiered approach to assess the adversity ofchemicals on vertebrates, and to link it with an estrogenic (E), androgenic (A), thyroid hormone(T), or steroidogenesis-related (S) mode of action (the so-called EATS modalities). The evidence
is first assembled by using a literature review and weight of evidence approach. Then, the EATSmediated adversity and the endocrine activity are assessed. The detailed methodology ispresented in the following sections.
A literature review was conducted up to May 2023. The literature review on TPhP endocrine
disruptive properties was focused on fish, amphibian and in vitro mechanistic studies. The
literature search was performed in Scopus database.
A single concept strategy search was applied to retrieve all relevant information on TPhP byusing its Chemical Abstracts Service Registry Number (CAS No 115-86-6), scientific chemicalnames, and common names, as recommended in the EDC guidance (ECHA/EFSA, 2018).
Studies were included in the literature review based on their relevance when they met all of thefollowing criteria: a) peer-reviewed research articles or primary reports of research findings that
presented original data; b) endocrine activity or adversity assessed in in vitro, ex vivo, or in vivo
studies in vertebrate species; and c) English-language articles. The relevance filtering was first
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ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC
based on title and abstract screening, and second, on full-text screening. When checking title
and abstract was insufficient to decide if the paper was relevant and should be included in thereview, full-text screening was applied (e.g., TPhP not explicitly mentioned in the abstract). Two
distinct reviewers shared the two screening phases during the literature search and resolved any
conflicts or discrepancies by complementary full-text screening and by discussion.
Assessment of the evidence
The present analysis was performed in collaboration with the ANSES Thematic Working groupon Endocrine Disruptors (EDC-WG)3. The studies were retrieved from the literature review aswell as from the registration dossier and were considered on the basis of their relevance (seecriteria of selection based on relevance above), reliability and adequacy for the analysis. Studies
were qualitatively weighted based on expert judgement to produce a conclusion on the selected
adverse effects and their ED MoA.
Analysis of the results
The data were grouped into three categories in accordance with the OECD conceptual framework(OECD, 2018) and EU EDC guidance (ECHA/EFSA, 2018):
a) in vitro mechanistic parameters;b) in vivo mechanistic parameters;c) parameters providing information on adversity.
Based on the adverse effects identified, results were further integrated into lines of evidence,
defined as a “set of relevant information grouped to assess a hypothesis,” using a weight-ofevidence approach (ECHA/EFSA, 2018).

In vitro information indicative of endocrine activity
Eighteen studies gathered in the literature and included in the review provide in vitro mechanistic
information on the capacity of TPhP to interact with the endocrine system. Most of the studies
focus on TPhP estrogenic activity.
Table 10: Summary table of in vitro mechanistic data (chronologic order)
STUDIES INVESTIGATING ENDOCRINE DISRUPTION PROPERTIES OF TPhP

3

Methodology

Results (statistically significant)Reference

AR competitive binding
assay (18 to 20h)
(Rat AR protein)
Dose: 4.28 nM to
0.428 mM.

IC50: 1.50E-05 M
AR Relative binding affinity (RBA):
0.0205% (moderate binding)Fang et al., 2003

H295R cell bioassay(48h)
Dose: 0 – 0.001 – 0.01 –0.1 – 1 mg.l-1

Hormone synthesis
17β-estradiol (E2):
Testosterone (T):E2/T Ratio:

Liu et al., 2012

Dose: 0 – 0.01 – 0.1 –1 mg.l-1

Steroidogenic genes
CYP11A1:↑ at 1 mg.l-1
CYP11B2:↑ at 1 mg.l-1
CYP19A1:↑ at 1 mg.l-1↑ at 1 mg.l-1↑ at 1 mg.l-1↑ at 0.1 and 1 mg.l-1

https://www.anses.fr/en/content/endocrine-disruptors.

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ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC

HSD3β2:

no effect

SULT genes
SULT1E1↓ at 1 mg.l-1
SULT2A1↓ at 1 mg.l-1
MVLN cell binding
bioassay (72h)Dose: 0 – 0.001 – 0.01 –0.1 – 1 - 10 mg.l-1↓ binding affinity of E2 for hER (0.001 mg.l-1)

Cell-based transactivation
Assay

Kojima et al., 2013

CHO-K1 cells (24h)Dose: 0.10 to 30 µM.

Erα: REC20 = 4.9 µM (weak agonist)
Erβ: REC20 = 6.5 µM (weak agonist)
AR: RIC20 = 17 µM (weak antagonist)
GR: RIC20 = 15 µM (weak antagonist)

COS-7 cells (24h)Dose: 0.10 to 30 µM.

PPARγ: weak agonist (15% at concentration of 30 µM)
PXR: REC20 = 2.8 µM (agonist)

Dual Luciferase Reporter
Gene Assay / CHO-K1
cells (24h)Dose: 0.1 nM to 1 µM.

Erα: REC20 of 0.27 µM (↑ activation of Erα in a dosedependent manner)

Yeast two-hybrid assay(4h)
Dose: 0.1 nM to 1 µM.

Erα: REC20 of 0.65 µM (↑ activation of Erα in a dosedependent manner)

E-Screen Assay / MCF-7
cells (5 days)
Dose: 0.1 nM to 1 µM.

Maximum cell proliferation: 1 µM
Relative proliferative effect: 37.5%

Molecular Docking and
Molecular Dynamic
Simulation using
Discovery Studio
2.5/Ligand fit module.

Tight binding affinity for hERα in docking approach(agonist effect)

GH3 (rat pituitary) cellassay (48h)Dose (µg.l-1) : 0 – 1(0.0003 µM) – 10
(0.031 µM) – 100(0.31 µM)

Zhang et al., 2014

Kim et al., 2015
Tshβ
Trα
trβ
dio1
dio2

↑ at 0.31 µM↑ at 0.031 & 0.31 µM↑ at 0.31 µM↑ at 0.31 µM
no effect

nis
tg
tpo

↑ at 9.2 & 31 µM↓ at 3.1 µM↑ at 31 µM

FRTL-5 (rat thyroid
follicular) cell assay (24h)Dose (mg.l-1) : 0 – 1 (3.1µM) – 3 (9.2 µM) – 10
(31 µM)27 (95)

ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC

tshr
nkx2.1
pax8↓ at 3.1 µM↓ at 3.1 & 31 µM
no effect

Cell-based transcriptional
Assay

Kojima et al., 2016

CHO-K1 cells (24h)Dose: 0.1 to 30 µM

Erα: REC20 = 4.6 µM (weak agonist)
Erβ: REC20 = 7.3 µM (weak agonist)
AR: RIC20 = 11 µM (weak antagonist)
GR: RIC20 = 12 µM (weak antagonist)

COS-7 cells (24h)Dose: 1 to 30 µM

PPARγ: weak agonist (15% at concentration of 30 µM)
PXR: REC20 = 2.8 µM (agonist)

MCF-7 flow-cytometricproliferation assay (24h)Dose: 10 µM to 1 mM

Relative proliferative effect (RPE) = 87% (E2 100%)
EC20 = 88 µM (E2 8E-07 µM)

MA-10 mouse Leydig
tumour cells (48h)Dose: 1 to 100 µM

qRT-PCR(Expression of Key Genes Involved in Steroidogenesis)
Lhcgr:
no effect
Adcy3:
no effectStar:
no effect
TSPO:
no effect
Cyp11A1:
no effect
Hsd3β:
no effect

Krivoshiev et al.,2016

Schang et al., 2016

Progesterone production: no effectTransthyretin (TTR)
binding experiment (2h)Dose: approx. 6 nM to
32 µM

IC50: 1083 nM (significant binding ability to TTR
compared to T4 = IC50 of 291 nM)

Zhao et al., 2017

Yeast two-hybrid assay
with Japanese medaka
Erα (mERα)
Dose: approx. 0.1 to100 µM (4h)IC50 = 29 µM (antagonist activity)

Li et al., 2018

Yeast two-hybrid assay
with human Erα (4h)0.5 nM to 0.1µM.l-1

hERα: Antagonist activity (RIC20 = 2E-05 mol/L)compete with E2 for binding to ErαJi et al., 2020hERE: Agonist response (EC50 = 1.45E-05 mol/L) =Erα agonist to activate the ERE pathway
MVLN cell assay withhuman ERE
(72h)
0.1 nM to 20µM.l-1

Promotes cell proliferation = agonist activity (REC20 =5.01E-07 M/L, max 77.08%). This proliferative effectwas significantly inhibited when cells were pretreatedwith G15 (a GP30 inhibitor).

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ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC

E-SCREEN assay(proliferation MCF7)

Multiplexed reporter
assay (the Ecotox
FACTORIAL)
40 µM (24h)Activation of hPPARγ, mPPARγ, zPPARγ
Activation of Zebrafish ER2β, frogER1, turtleER1,chickenER1, hERα
Activation of chickenARNo effect on ZebrafishER1 and ER2α, frogER2, hERβ,
AR (fish, frog, turtle, human), fishTRα and TRβ,frogTRα, turtleTRα, hTRα and hTRβHG5LN cell assay (24h)Dose (µM): 0.1 to 10 µM

Transcriptional activation bioassay:
Activation of hPPARγ (EC50: nd; % max. activity:
28%)
Activation of mPPARγ (EC50: nd; % max. activity:
24%)
Activation of xPPARγ: (EC50: nd; % max. activity:
21%)
Activation of zfPPARγ (EC50 = 3.5 µM; % max.
activity: 104%)

Multiplexed reporter
assay (the Ecotox
FACTORIAL)
22.2 µM

Medvedev et al.,2020

Agonist activity on mPPARγ, hPPARγ, zPPARγ

Garoche et al.,2021

Houck et al., 2021

Reporter gene assay:
HEK293 cell assay (39h)with medaka estrogen
receptor 1(mEsr1)
HepG2 cell assay (39h)with medaka androgen
receptor β (mArβ)

mEsr1:
Agonist activity (EC50 = 9.7E-06 M).
No antagonist activity up to 1.0 E-05 M.

Kawashima et al.,
2021

mARβ:
No agonist or antagonist activity up to 1.0 E-05
M.

Molecular dynamics
analysis

TPhP disturbed conformational changes to affect thestructural stability of TRβ ligand binding domain leading
to potential disruption of the transcriptional activity ofTR.

Transcriptomic analysis
on L02 cells (HL-7702,
human normal liver cells)
for 24h.
20 and 200µM

Gene expression↑ THRB, RXRA, DIO2 and PIK3R3 (from 20 µM)↓ TP53, NOTCHI1 (from 20 µM)↓ AKT3, DIO2, THRA, SLCO1C1 and CASP9 (200 µM)↑ MDM2, BAD (200 µM)

Molecular docking(CDOCKER)

With ERα, TPhP formed a pi-sigma bond with
Arg394 as E2 which in addition bond to
His524 and Glu353.
With GPER, TPhP formed a pi-sigma bondwith Tyr142 while E2 formed hydrogenbonds with Tyr123 and Glu275, and Van der
Waals forces with Cys205. G1, a GPER

Wang et al., 2021

Ji et al., 2022

29 (95)

ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC

agonist, had a hydrogen bond and a pi-pibond with Gln138 and Phe208. The
CDOCKER interaction energy with TPhP was- 29.60 kcal/mol.

H295R cell assay (48h)Dose (µM) : 0.1 – 1 – 2
and 10MVLN (hERα +) cellassay (72h)Hormone levels:
E2
E2/T ratio
Progesterone
EstroneTestosterone

Transcriptional genes4
CYP17
CYP19
3β-HSD2
17β-HSD1
HMGR5
StAR6
SULT2A1
CYP11A1
17β-HSD4↑ at 2 and 10 µM↑ at 1, 2 and 10 µM↑ at 2 and 10 µM↑ at 1, 2 and 10 µM↑ at 1, 2 and 10 µM↑ at 10 µM↓ at 1, 2 and 10 µM
No sign. effectNo sign. effect

Receptor activationhERαAgonist activity

Dose (µM) : 0.1 – 0.2 –1 – 2 – 10 – 20
SKBR3 (ERα-, GPER+)
cell assay (24h)Dose (µM) : 0.1 – 1 and
10↑ at 1, 2 and 10 µM↑ at 1, 2 and 10 µM↑ at 2 and 10 µM↓ at 2 and 10 µM↓ at 1, 2 and 10 µM

EC50 = 1.2E-05 M

GPER-cAMP
activation- Interaction with GPER at 1.0E-05µM via cAMP formation- The agonist effects of TPhP on
cAMP production were significantly
inhibited when TPhP was coexposed with G15.
- The E2-control group responded
as expected.

ERα- SRC (steroid
receptor co-activators)
recruitment assay (1 h)Dose 0.01 μM to1000 µM.

ERα agonist interaction with SRC1–1:
EC50 of 2.93E-04 mol/LERα agonist interaction with SRC3–3:
EC50 of 1.30E-05 mol/L,No effect on the other 8 SRCs peptides

Molecular dynamics
simulation

Signs of TPhP binding to GPER leading to the change of
its structural conformation for a possible activation
Guan et al., 2022

Competitive binding
assay:

TPhP inhibits the binding of E2-F to GPER (probe) (IC504.2 µM)

Fold above 1.75 are reported in this table.
HMGR: 3-Hydroxy-3-methylglutaryl-coenzyme A reductase.
6
StAR: Steroidogenic acute regulatory protein.
4530 (95)

ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHCSKBR3 cells (10 min)Dose: 1 nM to 100 µM
Transcriptomic andproteomic analysis(SKBR3 cells)

64 genes were enriched for both mRNA and protein
after TPhP exposure. Among these overlaps, 52
DEGs7/DEPs8 were found to be up-regulated or downregulated together. Combined transcriptome andproteome analysis showed that TPhP could exert its
estrogen-like proliferation-promoting effect through theinteraction of the PI3K-Akt9 signalling pathway, MAPK10
signalling pathway, and EGFR11 signalling pathway,
which are downstream pathways mediated by GPER.

Estrogen pathway
Overall, 10 studies investigating the estrogenic activity of TPhP in vitro were identified andanalysed. The main results are summarised below and further discussed in the conclusion
section.
TPhP has been tested in different types of in vitro assay including binding assays, transactivationin reporter gene assays, regulated gene expression and G protein-coupled estrogen receptor(GPER) signalling pathways.
It appears that TPhP has agonist activity on human ERα and / or ERβ receptor anddecreases the binding affinity of E2 to ER (Liu et al., 2012; Kojima et al., 2013; Zhang etal., 2014; Kojima et al., 2016; Medvedev et al., 2020, Ji et al., 2022). The agonist activity isalso reported for the medaka ER1 (Kawashima et al., 2021) and for the zebrafish ER2β, frog ER1,
turtle ER1 and chicken ER1 (Medvedev et al., 2020). In some yeast two-hybrid assays withhuman and medaka ERα, an antagonist effect is shown indicating a competition between
TPhP and E2 for binding to ERα (Li et al., 2018; Ji et al., 2020). Zhang et al. (2014) also
demonstrated a binding affinity of TPhP to hERα with a docking approach. Moreover, TPhP can
induce the ERE pathway as demonstrated by a dose-response agonist effect in MVLN cells(transfected with the human ERE-luciferase reporter gene) (Ji et al., 2020). This estrogenic
activity is supported by proliferation assays on MCF-7 cells that show TPhP promotes cellproliferation (Zhang et al., 2014; Krivoshiev et al., 2016; Ji et al., 2020).
In Guan et al. (2022), the authors explored the effects of TPhP on the key downstream signalling
pathways mediated by the G-coupled protein ER (GPER) through combined transcriptome andproteome analysis. These relationships were also tested on a SKBR3 cell-based fluorescentcompetitive binding assay. The authors report that TPhP binds GPER with an IC50 of 4.2 µM.
This is confirmed by the E2-F displacement experiments. Combined transcriptome and proteome
analyses show that TPhP could exert its estrogen-like proliferation-promoting effectthrough the interaction of the PI3K-Akt signalling pathway, MAPK signalling pathway, and EGFR
signalling pathway, which are downstream pathways mediated by GPER.
In Ji et al. (2022), the interaction of TPhP with GPER was confirmed by molecular docking andtheir activation on GPER-mediated signalling was investigated in SKBR3 cells. In fact, the assay
on SBRK3 cells shows that TPhP can interact with GPER and activate cAMP formation,
which is one of the rapid signalling pathways induced by GPER activation. However, thisactivation is 10-times less efficient than the reaction induced by E2. This activation via GPER is

DEGs: differentially expressed genes.
DEPs: differentially expressed proteins.
9
PI3K-Akt: phosphoinositide 3-kinase/protein kinase B.
10
MAPK: mitogen-activated protein kinase.
11
EGFR: epidermal growth factor receptor.
7831 (95)

ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC
also confirmed by the results obtained with a TPhP co-exposure with the specific GPER30
inhibitor, G15, which inhibits the effects of TPhP in the assay.
Conclusion: The available in vitro information demonstrate the capacity of TPhP tocompetitively bind and activate nuclear ERα and ERβ estrogen receptors of several
vertebrate species including human, rat, mouse, fish, chicken, frog and turtle asevidenced by ER transactivation in reporter cell lines, increased promoter occupancy,
induction of ER-regulated gene expression and related physiological cell responses(e.g., increased cell proliferation). Two recent studies showed that TPhP could alsobind and activate GPER.
Androgen pathway
TPhP binding capacity to the androgen receptor (AR) has been investigated in the study by Fanget al. (2003). The results indicate a moderate binding of TPhP to the AR.
Weak AR antagonist effects were observed in hamster CHO-K1 cells (Kojima et al., 2013; 2016).
Conclusions: The data available indicate that TPhP can moderately bind to the AR andcould induce a weak anti androgenic effect in vertebrate cells.
Thyroid pathway
The expression of thyroid hormone-related gene expression was investigated in rat pituitary GH3
cells and in rat thyroid follicular FRTL-5 cells (Kim et al., 2015). In GH3 cells, TPhP exposure led
to an up-regulation of trα from the medium tested dose (0.031 µM) and of tshß, trβ and dio1
gene expression at the highest concentration tested (0.31 µM). In FRTL-5 cells, TPhP exposure
led to a down-regulation of tg and tshr expression at 3.1 µM, an up-regulation of nis (9.2 µM
and 31 µM), and tpo gene expression (31 µM).
Conclusions: The results on GH3 cells and FRTL-5 cells show that TPhP can interact
with thyroid hormone receptors in the rat pituitary or thyroid gland cells and could
thus lead to stimulation of the synthesis of thyroid hormones.
Steroidogenesis
The steroidogenesis activity of TPhP was assessed in human adrenal cortico-carcinoma (H295R)
cells (Liu et al., 2012; Ji et al., 2022). These two assays show that TPhP affects steroidogenesis
byincreasing
estrogenlevels(17ß-estradiol:
E2)
(see

Figure 3).

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ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC

Figure 3: Proposed mechanism framework of estrogen disrupting effects for TPhP from the publication of Ji et al.
(2022). Hormones in red indicates a significant increase of levels and blue indicates a significant decrease of levels (fromJi et al., 2022).

TPhP leads to an increase of E2 and E2/T ratio (Liu et al., 2012; Ji et al., 2022) and ofprogesterone (Ji et al., 2022) suggesting that TPhP has also an estrogenic activity throughan action on steroidogenesis. Ji et al. (2022) shows that TPhP can promote cholesterolsynthesis and its transfer through cellular membranes by a significant increase of HMGR andStAR gene expression levels respectively. TPhP also induces an increase of the expression of the
CYP genes (CYP11A1, CYP11B2, CYP19A1, CYP19) and 17β-HSD1 gene, which are linked tosteroidogenesis. TPhP up-regulated CYP11A1 gene expression, which then can result in an
increased conversion of cholesterol to pregnenolone. In addition, the up-regulated CYP17 gene
expression may cause more androstenedione synthesis, and up-regulated 3β-HSD2 will impact
the next step of the steroid synthesis. The authors hypothesised that the increased progesterone
levels noted in the study may result from the up-regulation of CYP11A1 and 3β-HSD2 gene
expression. An increased androstenedione synthesis due to CYP17 up-regulation gene expression
will then be followed by an increased conversion of testosterone and estrone to E2 via the upregulated CYP19 and 17β-HSD1 gene expression, which ultimately resulted in an increase of E2
concentrations (mechanisms confirmed in two other studies on 5 phthalates (DEP, MEP, BBzP,
MBzP and DiBP) (Sohn et al., 2016) and on the trichloroethylene (Tachachartvanich et al., 2018).
In Ji et al. (2022) a decrease of estrone is observed while CYP19 is upregulated and E2 level
increases. The reason given by the authors is that the conversion of estrone to E2 by 17β-HSD1
is stronger than the activity of CYP19 to estrone synthesis. Lastly, inhibition of SULT2A1
expression observed in the two H295R assay may lead to more dehydroepiandrosterone (DHEA),
participating in the production of E2 (Liu et al., 2012; Ji et al., 2022).
Conclusion: The two H295R assays performed with TPhP show that TPhP affects
steroidogenesis by increasing estrogen levels (17ß-estradiol) and increasing
expression of CYP19 and 3β-HSD2 genes involved in this pathway.
Other endocrine pathways
TPhP is shown to activate the transcription of human, mouse, zebrafish or Xenopus PPARγ(peroxisome proliferator-activated receptor) (Garoche et al., 2021). Transcriptional activation ofhuman, mouse and fish PPARγ is also demonstrated in a multiplexed reporter assay (Medvedev
et al. 2020; Houck et al., 2021). Kojima et al. (2013; 2016) also report agonist activity of TPhP
on human PPARγ (COS-7 cells).
33 (95)

ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHCIn Kojima et al. (2013; 2016) the authors investigated interactions with PXR receptor from COS7 cell-based reporter gene assay (to detect the agonist activity of chemicals against PXR). TPhP
showed PXR agonist activity (REC20 = 2.8 µM).

In vivo mechanistic data with regard to an endocrine activity
Table 11: Summary table of in vivo mechanistic data (chronologic order)
STUDIES INVESTIGATING ENDOCRINE DISRUPTION PROPERTIES OF TPhP
Methodology

Results (statistically significant change)

Remarks

Reference

Adult Zebrafish(Danio rerio) – 4
months old

Female Plasma sex hormone
17β-estradiol (E2)
↑ at 1 mg.l-1
Testosterone (T)No change
11-ketotestosterone (11- No changeKT)
E2/T Ratio
No change
E2/11-KT Ratio↑ at 1 mg.l-1

2 (reliablewithrestriction)

Liu et al.,2012

Nominalconcentration:
0.04 – 0.2 – 1
mg.l-1
No analyticalmeasurement
Solvent < 0.01%
DMSO (v/v)
Exposure of maleand female fish
separately
Replicates: 3 with
3 fish for each
replicate
Exposure
duration: 14 days
Photoperiod : 16h
light – 8h dark
Watertemperature:
24°C ± 2°C

Male Plasma sex hormone
17β-estradiol (E2)
Testosterone (T)11-ketotestosterone (11-KT)
E2/T Ratio
E2/11-KT Ratio↑ at 1 mg.l-1↓ at 1 mg.l-1↓ at 1 mg.l-1↑ at 1 mg.l-1↑ at 1 mg.l-1

Female Transcriptional genes
CYP1712
CYP19
VTG113↑ at 1 mg.l-1↑ at 1 mg.l-1↓ at 1 mg.l-1

Male Transcriptional genes
CYP17
CYP19
VTG1

Test
material:
TPhP
Purityunknown
Nonguideline/e
xploratory
study
Not GLP↑ at 1 mg.l-1↑ at 1 mg.l-1↑ at 0.04; 0.2 and1 mg.l-1

General toxicity
No significant mortality was observed (no details
provided).

Water pH: 7.2 to
7.8
Dissolved oxygen:> 7 mg.l-1
Exposure
medium: renewal
every other day

12
13

CYP17 and CYP19A were measured in gonad.
VTG 1 was measured in liver.

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ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC

Zebrafishembryos/larvae(Danio rerio) –4hpf
Nominalconcentration:
0.02 - 0.2 - 2
mg.l-1
Measuredconcentration:
48 hpf: 0.02 –0.19 – 1.80 mg.l-1
120 hpf: 0 – 0 –1.39 mg.l-1
Solvent : 0.01%
DMSO
Replicates: Four
separate wellswith 20 eggs
Exposure
duration: up to
120 hpf

Expression of mRNA for genes in six receptor (AhR,
PPARα, ER1, TRα, G and MR) centered gene networks14
AHRRB↑ at 0.02 mg.l-1only
CYP1A1↑ at 2 mg.l-1
NCOR2↑ at 2 mg.l-1
CYP1B1↑ at 2 mg.l-1
PPARα↑ at 2 mg.l-1
PPARGC1A↑ at 2 mg.l-1↑ at 2 mg.l-1
LPL
IL6↑ at 2 mg.l-1
PPARg↑ from 0.2 mg.l-1
ER2b↑ at 2 mg.l-1
TRα↑ at 2 mg.l-1
FUS↑ at 2 mg.l-1
GR↑ at 0.02 mg.l-1
RELA↑ at 2 mg.l-1
TGFb1↑ at 2 mg.l-1
HSP90AA1↑ at 2 mg.l-1↑ from 0.2 mg.l-1
11βHSD
EGFR↑ at 2 mg.l-1
MR↓ at 2 mg.l-1
HPSE↓ at 2 mg.l-1

2 (reliablewithrestriction)

Liu et al.,
2013a

Test
material:
TPhP
Purityunknown
Nonguideline/e
xploratory
study
Not GLP

General toxicity
Rates of hatching and survival were >90% and rate ofmalformation (spinal curvature, pericardial edema andyolk sac edema) was <5% in all exposure groups
including controls (no details provided).

Exposure
medium: renewedat 48 h only.
Adult Zebrafish(Danio rerio) –4/5 months old
Nominalconcentration: 0 –0.04 – 0.2 –1 mg.l-1
Measuredconcentration:
0h: 0.03 – 0.14 –0.89 mg.l-1
48h: <LOD <LOD – 0.38
mg.l-1(LOD = 0.12 µg.l1)
Solvent: DMSO
(0.005%)
Replicates: 6
replicates with six
pair of fish
Exposure
duration: 21 days
14

Female plasma sex hormone and VTG levels
E2 level↑ at 1 mg.l-1
E2/11-KT ratio level↑ at 1 mg.l-1
E2/T ratio level↑ at 0.2 and 1 mg.l-1
VTG↑ at 0.2 and 1 mg.l-1
Testosterone level↓ at 1 mg.l-1
11-KT↓ at 1 mg.l-1
Male plasma sex hormone and VTG levels
E2 level↑ at 0.2 mg.l-1
E2/11-KT level↑ at 0.2 mg.l-1
VTG level↑ at 1 mg.l-1
E2/T level↓ at 0.04 and 0.2 mg.l-1
Transcriptional genes in ovaries
LHR↑ at 1 mg.l-1
FSHR↑ at 1 mg.l-1
HMGRA↑ at 1 mg.l-1
STAR↑ at 0.2 mg.l-1
17βHSD↑ at 0.04 mg.l-1
CYP17A↑ at 1 mg.l-1
CYP19A↑ at 1 mg.l-1
HMGRB↓ at 1 mg.l-1

2 (reliablewithrestriction)

Liu et al.,
2013b

Test
material:
TPhP
Purity:99%
Equivalentor similar
to OECDTG 229Not GLP

Transcriptional genes in testis↑ at 0.2 mg.l-1
CYP11A
CYP17↑ at 1 mg.l-1
CYP19A↑ at 1 mg.l-1
LHR↓ at 0.04 mg.l-1

Only upregulated or downregulated gene are reported in this table.

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ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC

Photoperiod : 14h
light – 10h dark
Watertemperature:
27 °C ± 1 °C
Exposure
medium: renewedin every 48h.

HMGRA
STAR
17βHSD↓ at 1 mg.l-1↓ at 1 mg.l-1↓ at 0.2 mg.l-1

Female transcriptional genes in brain
GnRH2↓ at 1 mg.l-1
GnRH3↓ at 0.2 mg.l-1
GnRHR3↓ at 0.2 mg.l-1↑ at 1 mg.l-1
GnRHR4LHβ↑ at 0.2 mg.l-1
FSHβ↑ at 0.2 mg.l-1
CYP19B↑ at 0.2 mg.l-1
AR↑ at 0.2 mg.l-1
Erα↑ at 1 mg.l-1
ER2β1↑ at 1 mg.l-1
Male transcriptional genes in brain
GnRH2↑ at 0.04 mg.l-1
GnRH3↓ at 0.04 mg.l-1↑ at 0.04 mg.l-1
GnRHR2GnRHR3↑ at 1 mg.l-1
FSHβ↑ at 1 mg.l-1
LHβ↓ at 0.04 mg.l-1
CYP19B↑ at 1 mg.l-1
AR↓ at 1 mg.l-1
Erα↑ at 1 mg.l-1
ER2β1↑ at 0.2 mg.l-1
General toxicity
No mortality was observed in any of the experimental
concentrations during the exposure period (no details
provided).

Zebrafishembryos/larvae(Danio rerio) –3hpf
Nominalconcentration: 0 –40 – 200 – 500µg.l-1
No analyticalmeasurement
Solvent: DMSO
(0.005%)
Replicates: 6 pertreatment with100 larvae per
replicate
Exposure
duration: 7 days
Photoperiod : 14h
light – 10h dark
Watertemperature:

Hormone measurements in whole-body homogenate of
7 dpf Zebrafish larvae (100 larvae per replicate used)
T3 level↑ at 40, 200 and 500 µg.l-1↑ at 40, 200 and 500 µg.l-1
T4 level
Gene expression in whole-body homogenate of 7 dpf
Zebrafish larvae (20 larvae per replicate used)
ttr↑ at 40, 200 and 500 µg.l-1
trα↑ at 200 µg.l-1
dio1↑ at 500 µg.l-1
nis↑ at 200 and 500 µg.l-1
tg↑ at 200 and 500 µg.l-1
ugt1ab↑ at 200 and 500 µg.l-1
crh↓ at 500 µg.l-1
trβ↓ at 500 µg.l-1
tshβ
No changetshr
No change
nkx2.1
No change
pax8
No change
Dio2
No change

2 (reliablewithrestriction)

Kim et al.,
2015

Test
material:
TPhP
Purity:99%
Nonguideline/e
xploratory
study
Not GLP

General toxicity
Larval survival at 7d: 91.8% (control) – 92.5% (40 µg.l1) – 92% (200 µg.l-1) – 88.7% (500 µg.l-1).
Body weight were not affected by the experimental
concentrations.

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ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC

25°C ± 1°C
Exposure
medium: 50%
renewed daily.
Larvae, juvenile
and adult
Zebrafish (Danio rerio) – 4hpf
Nominalconcentration:0 – 0.005 – 0.05
– 0.5 mg.l-1
Measuredconcentration:
24h: 0.0008 –0.0055 – 0.131
mg.l-1
48h: < LOD - <LOD – 0.011 mg.l1(LOD = 0.12 µg.l1)
Solvent: DMSO
(0.005%)
Replicates:3 pertreatment with
100 embryos per
replicate
Exposure
duration:
120 days
Photoperiod : 14h
light – 10h dark
Watertemperature:
27°C ± 1 °C
Water pH: 7.2 to
7.8
Dissolved oxygen:> 7 mg.l-1
Exposure
medium: 50%
renewed every
48h.

Female plasma sex hormone↑ at 0.005 and 0.5 mg.l-1
E2 level↑ at 0.5 mg.l-1
Cortisol level↓ at 0.5 mg.l-1
11-KT level↑ at 0.5 mg.l-1
E2/11-KT ratio level↑ at 0.5 mg.l-1
T3 level↑ at 0.5 mg.l-1
T4 level
Male plasma sex hormone
E2 level
Cortisol level11-KT levelE2/11-KT level
T3 level
T4 level↑ at 0.005 mg.l-1↓ from 0.05 mg.l-1↓ from 0.005 mg.l-1↑ from 0.005 mg.l-1
No change
No change

2 (reliablewithrestriction)

Liu et al.,
2016

Test
material:
TPhP
Purity:99%
Nonguideline/e
xploratory
Not GLP

Female transcriptional genes in brain
FSHβ↑ at 0.05 and 0.5 mg.l-1
LHβ↑ at 0.005 and 0.5 mg.l-1
GnRH3↑ at 0.005 and 0.5 mg.l-1
Erα↑ at 0.5 mg.l-1↑ at 0.5 mg.l-1
pomc
mr↑ at 0.5 mg.l-1
trhr2↑ at 0.5 mg.l-1
Male transcriptional genes in brain
LHβ↑ at 0.5 mg.l-1
GnRH3↓ at 0.5 mg.l-1
pomc↑ at 0.5 mg.l-1
trh↓ at 0.5 mg.l-1
FSHβ
No changeErαNo change
Transcriptional genes in ovaries↑ at 0.5 mg.l-1
LHR
STAR↑ at 0.05 and 0.5 mg.l-1
CYP19A↑ at 0.5 mg.l-1
CYP17
No change
FSHR
No change
3β-HSDNo change
17β-HSDNo change
Transcriptional genes in testisSTAR↓ at 0.5 mg.l-1
CYP17↓ at 0.5 mg.l-1
FSHR↑ at 0.005 mg.l-1
LHR↑ at 0.5 mg.l-1↑ at 0.5 mg.l-1
3β-HSD
17β-HSD↑ at 0.050 mg.l-1
CYP19ANo change
General toxicity
None of the test concentrations caused any distinct
malformation, and there were no significant differences

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ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC

in mortality among treatment groups during theexposure duration (no details provided).
Japanese medaka
transgenic(Oryzias latipes) –o dph – Only male

2 (reliablewithrestriction)
Testmaterial:
TPhP

Nominalconcentration: 1.6– 8 – 40 µg.l-1
Measuredconcentration:
0.134 – 0.299 –1.43 µg.l-1

Purity ≥95%
Nonguideline/e
xploratory

Solvent: DMSO
(0.001%)

Not GLP
Replicates: 2 pertreatment with 50
larvae per
replicate
Exposure
duration: 100days

17β-E2 level
T level11-KT level↑ at 0.29 and 1.43 µg.l-1↓ at 1.43 µg.l-1↓ at 1.43 µg.l-1

Li et al.,
2018

General toxicity
No data on systemic toxicity provided.

Photoperiod : 16h
light – 8h dark
Watertemperature:
25°C ± 1 °C
Water pH: 7.7 ±0.2
Dissolved oxygen:> 7.8 ± 0.3 mg.l-1
Number of tested
fish: 16 males
Exposure
medium: flowthrough system
Japanese medaka
transgenic(Oryzias latipes) –only females
Solvent: DMSO
(0.001%)
Photoperiod : 16h
light – 8h dark

Short-term exposure: 21 days
Female plasma sex hormone↑ at 1725 ng.l-1
17β-E2 level↑ at 1725 ng.l-1
Testosterone level
Female transcriptional genes in liver↓ at 1725 ng.l-1
VTG2

2 (reliablewithrestriction)
Testmaterial:
TPhP

Li et al.,2019b

Purity ≥95%

Watertemperature:
25°C ± 1 °C

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ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC

Water pH: 7.7±0.2
Dissolved oxygen:> 7.8 ± 0.3 mg.l-1
Exposure
medium: flowthrough system
Short-term
exposure: adultexposed during
21 daysLong-term exposure: 100 days
Female plasma sex hormone↓ at 1773 ng.l-1
17β-E2 level↓ from 131 ng.l-1
Testosterone level↓ from 131 ng.l-1
11-KT level

Nonguideline/e
xploratory
Not GLP

Female transcriptional genes in liver↓ from 131 ng.l-1
VTG1↓ from 363 ng.l-1
VTG2
General toxicity
No data on systemic toxicity provided.

Nominalconcentration: 1.6– 8 – 40 µg.l-1
Measuredconcentration:
243 – 589 –1725ng.l-1
Replicates: 2 pertreatment with 8
adult female per
replicateLong-term
exposure: larvae
exposed during
100 days
Nominalconcentration: 1.6– 8 – 40 µg.l-1
Measuredconcentration:
131 – 363 –1773 ng.l-1
Replicates: 2 pertreatment with 25
adult female per
replicateZebrafish adult(Danio rerio)NominalConcentration: 0– 0.04 – 0.2 – 1
mg.l-1
Measuredconcentration:

Female plasma sex hormone↑ at 1 mg.l-1
T4 level
T3 level↑ at 1 mg.l-1

2 (reliablewithrestriction)

Male plasma sex hormone
T4 level
T3 levelTestmaterial:
TPhP↓ at 1 mg.l-1↓ at 1 mg.l-1

Female transcriptional genes in brain
crh↓ at 1 mg.l-1

Liu et al.,2019

Purity 99%

39 (95)

ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC

0h: 0.03 – 0.15 –0.87 mg.l-1
48h: < LOD - <LOD – 0.42 mg.l-1(LOD = 0.12 µg.l1)
Solvent: DMSO
(0.005%)
Replicates:
For males: 3
replicates with 5
males perreplicate.
For females: 3
replicates with 5
females per
replicate.
exposure
duration: 14 days
Photoperiod : 14h
light – 10h dark
Water pH: 7.2-7.8
Dissolved oxygen:> 7 mg.l-1

tshβ
trα
trβ

↓ at 1 mg.l-1↑ at 0.2 and 1 mg.l-1↑ at 0.2 and 1 mg.l-1

Male transcriptional genes in brain
crh↑ at 0.2 and 1 mg.l-1
tshβ↑ at 1 mg.l-1
trβ↑ at 0.2 and 1 mg.l-1
trα
No change

Nonguideline/e
xploratory
study
Not GLP

Female transcriptional genes in thyroidtrβ↓ at 1 mg.l-1
trα↑ at 1 mg.l-1
dio2↑ at 1 mg.l-1
tg
No changetpo
No change
Dio1
No change
Male transcriptional genes in thyroidtrβ↓ at 0.2 and 1 mg.l-1
dio1↓ at 0.2 and 1 mg.l-1
dio2↓ at 0.2 and 1 mg.l-1
trα
No changetg
No changetpo
No change
General toxicity
No mortality was observed over the exposure period (nodetails provided).

Exposure
medium: renewedat least threetimes per week
Zebrafish adult(Danio rerio): 5month adultSub-chronic
toxicity (10% of
LC50)
Nominalconcentration: 80µg.l-1
No analyticalmeasurementTPh

P
Solvent: DMSO
(0.01%)

2 (reliablewithrestriction)
Sex hormone and VTG levels in female whole-body
homogenates
E2/T ratio level↓ at 80 µg.l-1
VTG level↓ at 80 µg.l-1
E2
No change
T
No change
Sex hormone and VTG levels in male whole-body
homogenates
E2/T ratio level
No change
VTG
No change
E2
No change
T
No changeTestmaterial:
TPhP
Purity 99%
Nonguideline/e
xploratory
study

He et al.,2021

Not GLP

General toxicity
No data on systemic toxicity provided.

Replicates:10
males and 10

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ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC

females exposed
separately
Exposure
duration: 21 days
Water
temperature:
25°C ± 1 °C
Photoperiod : 14h
light – 10h dark
Water pH: 7.2 ±0.2
Dissolved oxygen:> 6.8 ± 0.2 mg.l-1

Exposure
medium: renewed
every dayJapanese medaka(Oryzias latipes) –16± 2 weeks old

2 (reliablewithrestriction)

OECD 229

Testmaterial:
TPhP

Nominalconcentration(µg.l-1): 20 – 64 –200 – 640
Measuredconcentration(µg.l-1): 2.13 –7.19 – 17.1 –44.9

Purity>97%

Female plasma VTG level↓ at 7.19, 17.1 and 44.9 µg.lVTG level
1(LOQ = 0.03 µg.l1)

Replicates: 4
replicates pertreatment with 3
males and 3
females per
replicate

General toxicity
4.2% of mortality at 44.9 µg.l-1 (1 female). No
remarkable abnormal response (feeding activity,
equilibrium, swimming…). No statistical analysis forlength and weight parameters.

Accordingto OECDTG 229Not GLP
Kawashima
et al., 2021

Exposure
duration: 21 days
Water
temperature:
25°C ± 2 °C
Photoperiod : 16h
light – 8h dark

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ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC

Water pH: 6.5-8.5
Dissolved oxygen:> than 60% of air
saturation

Exposure
medium: Flowthrough (5times/day)
Zebrafishembryos (Daniorerio) – 72hpf
Nominalconcentrations:0.32 – 0.97 – 3.2– 9.8 mg.l-1
Solvent: DMSO
(0.1% v/v)
Replicates :20 embryos pertreatment
Exposure
duration : 24h
OECD 234 (Fishsexualdevelopment test
– FSDT)
Zebrafish (Daniorerio)
Nominalconcentration: 1,
3.2, 10, 32, 100µg.l-1
Measuredconcentration:
1.11 – 3.01 –7.76 – 33.3 –76.8 µg.l-1 (TPhP
in 1L acetone)

Transcriptional genes
trα
ttr
tshβ
dio1
dio2

2 (reliablewithrestriction)↓ at 3.2 mg.l-1↓ at 3.2 mg.l-1↓ at 9.8 mg.l-1↓ at 3.2 mg.l-1↑ at 3.2 mg.l-1

General toxicity
Significant concentration-dependent decrease in bodylength at 0.97, 3.2 and 9.8 mg.l-1 (n=10 embryos pertreatment).
Mortality > 50% at 9.8 mg.l-1. No significant mortality
observed for other concentrations (no details provided).

Testmaterial:
TPhP
Purity >98%

Lee et al.,2022

Nonguideline/e
xploratory
study
Not GLP

Female plasma sex hormone and VTG level*↑ at 33.3 µg.l-1
VTG level
17β-E2 level↑ at 3.01, 7.76, 33.3
and 76.8 µg.l-1

Male plasma sex hormone and VTG level*↑ at 33.3 µg.l-1
VTG level↑ at 3.01 µg.l-1
11-KT level

General toxicity
Survival rates were 88% (control group) - 84% (1.11µg.l-1); 80% (3.01 µg.l-1); 77% (7.76 µg.l-1); 63%(33.3 µg.l-1) - 51% (76.8 µg.l-1)

2 (reliablewithrestriction)
Testmaterial:
TPhP
Purity >99%OECD TG
234
GLP

Unpublishe
d studyreport,2022

Replicates: 4
replicates perconcentration with *Statistical unit: all individual measurements per concentration (not the
30 fertilised eggs
mean of replicates per concentration in contrast to analysis provided in
per replicate.
the study report).
Watertemperature:
27°C ± 2 °C

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ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC

Photoperiod : 12h
light – 12h dark
Water pH: 7.6-7.8
Dissolved oxygen:> than 60% of air
saturation

Exposure
duration: 73 days
Exposure
medium: flowthrough system
Zebrafish embryo(Danio rerio) –(0-2 hpf)

2 (reliablewithrestriction)

Nominalconcentration: 0 –20 – 50 – 100 –500 and 1000
µg.l-1

Testmaterial:
TPhP

No analyticalmeasurement
Solvent: DMSO
(0.005% v/v)
Watertemperature:
28°C ± 1 °C
Photoperiod : 14h
light - 10h dark

Replicates: 3
replicates pertreatment with
40 embryos per
replicate
Exposure
duration: 7 days
Exposure
medium: 90 %fresh exposure
solution changed
dailyTranscriptional genes in whole body (96 hpf)
↓ at 20 µg.l-1
BCL2A↓ at 500 µg.l-1
CASP9↓ at 1000 µg.l-1
MBPA↓ at 1000 µg.l-1
SYN2A↓ at 1000 µg.l-1
FOXO3A↓ at 500 and 1000 µg.l-1
PAX6A↑ at 50 µg.l-1
ALDH1A2↓ at 1000 µg.l-1
RDH1↓ at 1000 µg.l-1
CRABP1A↓ at 1000 µg.l-1
RBP2A↓ at 1000 µg.l-1
RARAA↓ at 500 and 1000 µg.l-1
CYP26A1↓ at 100, 500 and 1000
RBP5µg.l-1

Purity >98%
Nonguideline/e
xploratory
study
Not GLP

Zhang etal., 2023

Transcriptional genes in whole body (7 dpf)
↑ at 100 and 500 µg.l-1
ALDH1A2↑ at 20 and 50 µg.l-1
MBPA↑ at 500 µg.l-1
RDH1↑ at 20 µg.l-1
RBP2A
General toxicity
From 120 hpf, the survival rate of zebrafish larvae wassignificantly decreased at 500 μg.l-1 and 1000 μg.l-1.
Survival rate: 30% at 500 μg.l-1 and 70% at 1000 μg.l-1
7 dpf.

Measurement at
96 hpf and 7 dpf

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ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC
Thyroid pathway
Clear sex-dependent alterations in plasma TH levels were observed in zebrafish with an increaseof the plasmatic concentration of T3 and T4 in females (Liu et al., 2016; Liu et al., 2019) and adecrease or no change of the plasmatic concentration of T3 and T4 in males (Liu et al., 2016;2019). Kim et al. (2015) also reported an increase of the whole body homogenate concentrationof T3 and T4 in zebrafish embryos/larvae.
EAS modalities
The in vivo mechanistic effects described in the literature indicate that TPhP has EAS endocrine
activity.
In Liu et al. (2012), the transcription of steroidogenic genes (CYP17 and CYP19A) was measured
in adult Zebrafish gonads. Exposure of male and female to 1 mg.l-1 of TPhP significantly increased
CYP17 and CYP19A gene transcription. In Liu et al. (2013b), exposure to TPhP led to significant
transcriptional changes in both fish testes and ovaries. In testes, transcription levels of HMGRA,
StAR, and 17β-HSD were significantly down-regulated, while transcriptions of CYP11A, CYP17,
and CYP19A were significantly up-regulated. In ovaries, the major steroidogenic genes, including
HMGRA, StAR, 17β-HSD, CYP17A, and CYP19A, were significantly up-regulated by exposure to
TPhP.
Several studies demonstrated a significant increase of plasmatic concentrations of E2 and E2/11KT ratio in both sexes of zebrafish at the top dose (Liu et al., 2012; 2013b; 2016). An increaseof E2 level was also noted in female zebrafish (Unpublished study report, 2022), male medaka(Li et al., 2018) and female medaka (Li et al., 2019b). Nevertheless, in one study (Li et al.,2019b) carried out on Japanese medaka, a decrease of E2 was noted after 100-day exposure offemale larvae (age of fish at exposure initiation), while an increase of E2 was observed after 21days exposure of adult females (age of fish at exposure initiation). Finally in one study (He etal., 2021), no change in E2 level in male or female whole body homogenates was noted.
Concerning testosterone, an increase in the plasma level could be observed after a short term
exposure in female medaka plasma, while a decrease was noted after a long-term exposure (Liet al., 2019b). In He et al. (2021), no change was recorded in female and male fishhomogenates. A decrease of testosterone was observed in male medaka and zebrafish (Li et al.,2018; Liu et al., 2012) and in female zebrafish (Liu et al., 2013b). Decreased levels of thehormone 11-ketotestosterone (11-KT) were recorded in female fish after exposure to TPhP (Liuet al., 2013b; Liu et al., 2016; Li et al., 2018). The variation in 11-KT for males also followed adecreasing trend (Liu et al., 2012; Liu et al., 2016; Liu et al., 2018), except in the unpublishedstudy report (2022), where the 11-KT increased in male fish. Moreover, E2/11-KT ratio increasedin females and males (Liu et al., 2012; Liu et al., 2016; Li et al., 2018; Liu et al., 2013b). This
trend could not be verified in the unpublished study report (2022), as E2 levels were notmeasured in males and consequently the E2/11KT ratio could not be calculated.
In the same way, an activity on the E2/T ratio was noted, but the direction of variation was notestablished, with an increase of E2/T reported in some studies for males (Liu et al., 2012) or
females (Liu et al., 2013b), and a decrease or no change reported in others for females (Liu etal., 2012) or males (Liu et al., 2013b) or both (He et al., 2021).
The observations of VTG concentrations also suggest an estrogenic activity of TPhP in female
and male zebrafish, with increased plasmatic concentrations of VTG reported in Liu et al. (2013b)
and the Unpublished study report (2022). Contradictory results are also reported with decreased
or no changed concentrations in plasma VTG of female medaka (Kawashima et al., 2021) or inwhole-body homogenate of male and female zebrafish (He et al., 2021). VTG1 gene expression
in liver is increased in male zebrafish in one assay (Liu et al., 2012), while the expression in liverof VTG1 and 2 are decreased in other assays (Liu et al., 2012, for females; Li et al., 2019b).
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ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC
Conclusion: Transcription levels of key genes involved in the steroidogenesis was
significantly upregulated by exposure to TPhP in several studies. Significant alterationof plasmatic concentrations of E2, and E2/T ratio (more specific observation infemales) and E2/11-KT ratio (more specific observation in males) could result fromthis modification in the steroidogenesis pathway. These different perturbations ofcirculating steroid concentrations could depend on the fish developmental stage,
species and tested concentrations. The observations of VTG concentrations, that wereconsistent with perturbation of E2 concentrations, suggested an EAS activity of TPhPin female and male zebrafish, with altered plasmatic concentration of VTG.

Other endocrine pathways
In Zhang et al. (2023), the authors investigated the effects of TPhP exposure on transcriptional
levels in zebrafish at different developmental stages namely 96 hpf and 7 dpf. The expression
level of different genes were affected in zebrafish and 96 hpf larvae were shown to be moresensitive than 7 dpf larvae. The expression of apoptotic-related genes (bcl2a and Casp9) was
down-regulated in 96 hpf zebrafish at the concentration of 20 µg.l-1 and at 1000 µg.l-1
respectively. The expression of neurodevelopmental-related genes (pax6a and mbpa, syn2a,
foxo3a) was also down-regulated from 500 µg.l-1 and at 1000 µg.l-1 respectively.
Regarding the retinoic acid (RA) metabolic-related gene expression, at 96 hpf, the expression of
aldh1a2 was significantly up-regulated in 50 µg.l-1 TPhP. Exposure to 1000 μg/L TPhP
significantly downregulated the expressions of rdh1, crabp1a, rbp2a and raraa. Furthermore,
the expression of cyp26a1 was significantly decreased in the 500 and 1000 µg.l-1 TPhP treatedgroups. The expression of rbp5 was also decreased in 100, 500 and 1000 µg.l-1 TPhP treatedgroups. Several genes in the RA metabolic pathway were also examined in zebrafish larvae at 7
dpf. The expression of aldh1a2 was significantly up-regulated in 100 and 500 µg.l-1 TPhP treatedgroups. And exposure to 500 µg.l-1 TPhP significantly increased the expressions of rdh1.
Exposure to 20, 50, 100 and 500 µg.l-1 TPhP significantly up-regulated the transcriptions of
rbp2a.
Although not extensively detailed here, it is also important to note that TPhP was previously
shown to interact with the retinoic acid receptor (RAR). TPhP exhibits weak RARα antagonist
activity (Jia et al., 2022; Isales et al., 2015), which could lead to ocular and cardiovascularmalformations. The RAR signaling pathway is essential for reproduction and embryonicdevelopment. Indeed, genetic studies in zebrafish embryos that are deficient in RA-generating
enzymes or RARs revealed that retinoic acid (RA) signalling regulates development of manyorgans and tissues, including the body axis, spinal cord, forelimb buds, skeleton, heart, eye,pancreas, lung and spermatogenesis (Ghyselinck et al. 2019; Clagett-Dame and DeLuca, 2002).
There are many additional functions for RA that are supported by in vivo genetic loss-of-function
studies in zebrafish. Further studies are needed to identify the key genes regulated by RA
signalling. The retinoid signaling pathway is an endocrine pathway considered to be susceptible
to environmental endocrine disruption (OECD, 2021) and possibly related to endocrine effects
on development.

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ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC

In vivo adverse effect data with regard to an endocrine mode of action
Table 12: Summary table of in vivo adverse effect data (chronologic order)
STUDIES INVESTIGATING ENDOCRINE DISRUPTION PROPERTIES OF TPhP
Methodology

Results (statistically significant change)

Remarks

Reference

Adult Zebrafish(Danio rerio) –4/5 months old
Nominalconcentration:0 – 0.04 – 0.2 –1 mg.l-1
No analyticalmeasurement
Measuredconcentration:
0h: 0.03 – 0.14
– 0.89 mg.l-1
48h: <LOD <LOD – 0.38
mg.l-1(LOD = 0.12
µg.l-1)
Solvent: DMSO
(0.005%)
Replicates: 6
replicates with
six pair of fish
Exposure
duration: 21
days

Female Zebrafish
Cumulative Egg
numberSpawning event
Hatchability
Fertilisation successHepato-somatic
index (HSI)
Gonado-somatic
index
(GSI)

↓ at 0.2 and 1 mg.l-1↓ at 0.2 and 1 mg.l-1↓ at 0.2 and 1 mg.l-1
No change

2 (reliable withrestriction)
test material: TPhPNo change
No change

Purity: 99%

Liu et al.,2013b

Equivalent or similar
to OECD TG 229

General toxicity
No mortality was observed in any of theexperimental concentrations during the exposure
period (no details provided).

Not GLP

Photoperiod :14h light - 10h
dark
Water
temperature:
27 °C ± 1 °C

Exposure
medium:renewed inevery 48h.
Zebrafishembryos/larvae(Danio rerio) –3hpf
Nominalconcentration:

Malformation rate

↑ at 500 µg.l-1

2 (reliable withrestriction)

Kim et al.,2015

Test material: TPhP
General toxicity

Purity: 99%

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ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC

0 – 40 – 200 –500 µg.l-1
No analyticalmeasurement

Larval survival at 7d: 91.8% (control) – 92.5% (40µg.l-1) – 92% (200 µg.l-1) – 88.7% (500 µg.l-1).
Body weight were not affected by the experimental
concentrations.

Nonguideline/exploratory
Not GLP

Solvent: DMSO
(0.005%)
Replicates: 6
per treatment
with 100 larvae
per replicate
Exposure
duration: 7
days
Photoperiod :14h light - 10h
dark
Water
temperature:
25°C ± 1°C
Exposure
medium: 50%
renewed daily.
Larvae, juvenile
and adult
Zebrafish(Danio rerio)Nominalconcentration:0 – 0.005 –0.05 – 0.5 mg.l1

Female Zebrafish
Gonadosomatic
index (GSI)
Hepato-somatic
index (HSI)
Condition factor (CF)↓ at 0.005 and
0.5 mg.l-1
No change

No change
Measuredconcentration:
First 24h:
Male Zebrafish
0.0008 - 0.0055- 0.131 mg.l-1
Gonadosomatic
No change
First 48h: <index (GSI)
LOD - < LOD Hepato-somatic
No change
0.011 mg.l-1
index (HSI)
Condition factor (CF) ↓ at 0.5 mg.l-1
Solvent: DMSO
(0.005%)General toxicity
Replicates:3 per None of the test concentrations caused any distinct
treatment withmalformation, and there were no significant
100 embryos
differences in mortality among treatment groups
per replicate
during the exposure duration (no details provided).

2 (reliable withrestriction)
Test material: TPhP
Purity: 99%

Liu et al.,2016

Nonguideline/exploratory
Not GLP

Exposure
duration:
120 days

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ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC

Photoperiod :14h light - 10h
dark
Water
temperature:
27°C ± 1 °C
Water pH: 7.2
to 7.8
Dissolved
oxygen: > 7
mg.l-1
Exposure
medium: 50%
renewed every
48h.
Japanesemedakatransgenic(Oryzias latipes)– < 4hpf

2 (reliable withrestriction)

Nominalconcentration:
5, 25, 125, 625µg.l-1

Purity > 99%

No analyticalmeasurement

Not GLP

Solvent: DMSO
(0.01%)
Replicates: 3
replicates perconcentration
Water
temperature:
25°C ± 1 °C

Test material: TPhP

Nonguideline/exploratory

Hatchability (%) at 96hTime of hatching at 96h
General toxicity (14 d)Heart rates
Body length
Gross abnormality
Mortality↓ at 625 µg.l-1↑ at 625 µg.l-1
Sun et al.,2016

↓ at 125 and 625 µg.l-1↓ at 125 and 625 µg.l-1↑ at 625 µg.l-1
No information given

Photoperiod :14h light - 10h
dark
Exposure
duration: 14 d
Exposure
medium:renewed daily(at least 90%)
Japanese
Male Medaka:
medakatransgenic(Oryzias latipes)
Gonadal intersex– 0 dph - Only
incidencemale

2 (reliable withrestriction)↑ at 1.43 µg.l-1

Test material: TPhP

Li et al.,2018

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ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC

Nominalconcentration:
1.6 – 8 – 40
µg.l-1
Measuredconcentration:
0.134 – 0.299 –1.43 µg.l-1
Solvent: DMSO
(0.001%)

Abnormal chasing
behaviours↑ at 0.29 and 1.43
µg.l-1

Courtship behaviour↑ at 0.29 µg.l-1

Chasing time

↑ at 0.29 µg.l-1

Successful matingrate↓ at 1.43 µg.l-1

Purity ≥ 95%
Nonguideline/exploratory
Not GLP

General toxicity
No data on systemic toxicity provided.

Replicates: 2
per treatment
with 50 larvae
per replicate
Exposure
duration: 100
days
Photoperiod :16h light - 8h
dark
Water
temperature:
25°C ± 1 °C
Water pH: 7.7± 0.2
Dissolved
oxygen: > 7.8± 0.3 mg.l-1
Number oftested fish: 16
males
Exposure
medium: flowthrough system
JapanesemedakaFemale medaka:transgenic(Oryzias latipes)– only females
Long-term exposure: 100 days
Solvent: DMSO
↓ at 0.13, 0.36 and(0.001%)
Number of mature
oocyte1.77 µg.l-1
Photoperiod :↓ at 0.36 and 1.77
Egg production
16h light - 8h
µg.l-1dark
Water
temperature:
25°C ± 1 °C

General toxicity
No data on systemic toxicity provided

2 (reliable withrestriction)
Test material: TPhP
Purity ≥ 95%

Li et al.,2019b

Nonguideline/exploratory
Not GLP

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ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC

Water pH: 7.7±0.2
Dissolved
oxygen: > 7.8± 0.3 mg.l-1
Exposure
medium: flowthrough system

Long-term
exposure:larvae exposed
during 100 days
Nominalconcentration:
1.6 – 8 – 40
µg.l-1
Measuredconcentration:
131 – 363 –1773 ng.l-1
Replicates: 2
per treatment
with 25 adultfemale per
replicateJapanesemedaka(Oryzias latipes)– 16± 2 weeks
oldSecondary sex
characteristics

No change

Test material: TPhPOECD 229

Egg number (female)↓ at 44.9 µg.l-1

Purity >97%

Nominalconcentration(µg.l-1): 20 – 64
– 200 – 640

Fertilised egg number

Measuredconcentration(µg.l-1): 2.13 –7.19 – 17.1 –44.9
Replicates: 4
replicates pertreatment with
3 males and 3
females per
replicate2 (reliable withrestriction)

According to OECD
TG 229Fertility rate

↓ at 44.9 µg.l-1

HSI (male)No change

Not GLP
Kawashima
et al., 2021

↑ at 7.19, 17.1, 44.9
µg.l-1
General toxicity
4.2% of mortality at 44.9 µg.l-1 (1 female). No
remarkable abnormal response (feeding activity,
equilibrium, swimming…). No statistical analysis forlength and weight parameters

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ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC

Exposure
duration: 21
days
Water
temperature:
25°C ± 2 °C
Photoperiod :16h light - 8h
dark
Water pH: 6.58.5
Dissolved
oxygen: > than
60% of air
saturation

Exposure
medium: Flowthrough (5times/day)

Zebrafish adult(Danio rerio):
5-month adultSub-chronic
toxicity (10% of
LC50)

Female Zebrafish:
Hepato-somatic index(HSI)
Gonado-somatic index(GSI)
Ovary maturation
stagesNominalconcentration:
80 µg.l-1
No analyticalmeasurement
Solvent: DMSO
(0.01%)

Replicates:10
males and 10
females
exposedseparately
Exposure
duration: 21
days
Water
temperature:
25°C ± 1 °C

2 (reliable withrestriction)↑ at 80 µg.l-1↓ at 80 µg.l-1(oocytes less mature
with more oocytes at
the first two stages(perinuclear andcorticolar alveolar
oocytes). Structure ofoocytes more
irregular. Inhibition of
the ovariandevelopment.

Male Zebrafish:
Hepato-somatic index(HSI)
Gonado-somatic index(GSI)
Spermatocyte
maturation stages

Test material: TPhP

↑ at 80 µg.l-1
Purity 99%
Nonguideline/exploratory
Not GLPHe et al.,2021

↑ at 80 µg.l-1↓ at 80 µg.l-1↓ at 80 µg.l-1 (moreimmature
spermatocytes(spermatogonia andspermatocyte) andless mature
spermatocytes(spermatids andspermatozoa).

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ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC

Photoperiod :14h light - 10h
dark

Fecundity/fertility

Water pH: 7.2± 0.2
Dissolved
oxygen: > 6.8± 0.2 mg.l-1↓ at 80 µg.l-1 offertilisation rate,
cumulative eggs,
spawning, hatchingrate.

General toxicity
No data on systemic toxicity provided.

Exposure
medium:renewed every
day TPhP
Amphibiantadpoles (Ranazhenhaiensis)

2 (reliable withrestriction)
Test material: TPhP

Solvent: DMSO
(0.01%)

Purity > 99%

Nominalconcentration:
0.02 and 0.1
mg.l-1

Nonguideline/exploratory

No analyticalmeasurement

Non-standard
species

Not GLP

Developmental stages

Exposure
duration: 30
days↑ at 0.1 mg.l-1 (moreadvanceddevelopmental stage)

Lu et al.,2021

General toxicity
Replicates: 6
No death occurred in any treatment groups
replicatethroughout the exposure duration.
aquaria, 60
tadpoles in eachtreatment group
Stage GS26
(Gosner stage)
Water
temperature:
25°C ± 1°C
Photoperiod :12h light - 12h
darkAmphibians
tadpolesSurvival (30d)(Hoplobatrachus
Metamorphosis rates
rugulosus)(30d)↓ at 50 µg.l-1↓ at 50 µg.l-1

2 (reliable withrestriction)
Test material: TPhP

Chen et al.,2022

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ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC

Stage GS35
(Gosner stage)

Metamorphosis time
(30d)

Solvent: DMSO
(0.5 ‰ v/v)

NonGeneral toxicity
guideline/exploratory
Survival rate: 87.8% (control group) – 87.8%(solvent control group) – 76.7% (10 µg.l-1) – 66.7% Not GLP(50 µg.l-1) – 63.3% (100 µg.l-1) – 61.1% (200 µg.l-1)

Nominalconcentration:
0- solvent
control - 10 –50 – 100 – 200µg.l-1↓ at 100 µg.l-1

Purity > 99%

Exposure
duration: 30
days
Replicates: 3
replicates pertreatment with
30 tadpoles per
replicate
Water
temperature:
29°C ± 2 °C
Photoperiod :14h light - 10h
dark
Water pH: 7.2± 0.2
Dissolved
oxygen: > 5.78
± 0.3 ppm
Exposure
medium:renewed daily
OECD 234 (Fishsexualdevelopment
test – FSDT)
Zebrafish(Danio rerio)Nominalconcentration:
1, 3.2, 10, 32,
100 µg.l-1
Measuredconcentration:
1.11 – 3.01 –7.76 – 33.3 –76.8 µg.l-1(TPhP in 1L
acetone)2 (reliable withrestriction)
Sex ratio
No statistically significant change% of females: 64.8% (control) – 52.6% (1.11 µg.l-1)– 55.8% (3.01 µg.l-1); 59.1% (7.76 µg.l-1) – 67.5%(33.3 µg.l-1) – 65.4% (76.8 µg.l-1)
Note: high proportion of females in the control group(64.8%)

Test material: TPhP
Purity > 99%
OECD TG 234
GLP

Male Zebrafish:
Testis maturation
stagesUnpublishedstudy
report,2022↑ at 76.8 µg.l-1

General toxicity
Survival rates were 88% (control group) - 84%(1.11 µg.l-1); 80% (3.01 µg.l-1); 77% (7.76 µg.l-1);
63% (33.3 µg.l-1) - 51% (76.8 µg.l-1)

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ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC

Replicates: 4
replicates perconcentration
with 30
fertilised eggs
per replicate.
Water
temperature:
27°C ± 2 °C
Photoperiod :12h light - 12h
dark
Water pH: 7.67.8
Dissolved
oxygen: > than
60% of air
saturation

Exposure
duration: 73
days
Exposure
medium: flowthrough system
Zebrafishembryos/larvae(Danio rerio) –2hpf
Nominalconcentration:
8, 24, 72, 144µg.l-1
No analyticalmeasurement
Replicates: 3
replicates perconcentration
Water
temperature:
28°C ± 0.5 °C
Photoperiod :14h light - 10hdark

2 (reliable withrestriction)
Test material: TPhP
Purity > 99%
Hatching rates

General toxicity
Heart rates
Malformation rates
Survival rates
Body length↓ at 72 and 144 µg.l-1

Nonguideline/exploratory
Not GLP↓ at 72 and 144 µg.l-1↑ at 72 and 144 µg.l-1↓ at 144 µg.l-1↓ at 144 µg.l-1

Fan et al.,2022

Survival rates
86.5% (control group) – 86% (8 μg.l-1) - 84%(24 μg.l-1) – 82% (72 μg.l-1) – 80.5% (144 μg.l-1)

Exposure
duration: 120 h

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ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC

Exposure
medium:renewed daily
Zebrafishembryo (Daniorerio) – (0-2hpf)

2 (reliable withrestriction)
Test material: TPhP

Nominalconcentration:
0 – 20 – 50 –100 – 500 and1000 µg.l-1

Survival rate

No analyticalmeasurement
Solvent: DMSO
(0.005% v/v)
Watertemperature:
28°C ± 1 °C
Photoperiod :14h light - 10h
dark

Replicates: 3
replicates pertreatment with
40 embryos per
replicate↓ at 1000 µg.l-1 (6 dpf)
↓ at 500 µg.l-1 (7 dpf)

Purity > 98%

Hatching rate

↓ at 50 µg.l-1 at 48 hpf↓ at 500 µg.l-1 at 72 hpf

Nonguideline/exploratory

Heart rate

↑ at 100 and 1000 µg.l-1
at 48 hpf
↓ at 500 µg.l-1 at 96 hpf

Not GLP

Behaviour changes
Swimming distance
Movement trajectory
Number of apoptotic
cells in the head,
heart and intestine↑ at 20; 100; 500
and 1000 µg.l-1↑ at 20; 100; 500
and 1000 µg.l-1

Zhang etal., 2023↑ with the increase of
TPhP concentration

General toxicity
From 120 hpf, the survival rate of zebrafish larvae
was significantly decreased at 500 μg.l-1 and 1000
μg.l-1.

Exposure
duration: 7
days

Thyroid pathway
Lu et al. (2021) studied exposure of amphibian tadpoles (Zhenhai brown frog, Ranazhenhaiensis) throughout the developmental period to assess physiological and metabolic
impacts of TPhP on amphibian larvae. The exposure started with tadpoles at the Gosner stage(GS) 26. A total of 180 tadpoles were randomly selected and allocated to different aquaria, eachcontaining the solvent control (DMSO), 0.02 and 0.1 mg TPhP.l-1 (6 replicate aquaria, 60 tadpolesin each treated group). After 30-days exposure the tadpoles were euthanised. Snout-vent length(SVL), tail length (TL), body weight (BW) and developmental stage were recorded. No deathoccurred in any treated groups throughout the exposure duration. Tadpoles from 0.1 mg.l-1treated group had more advanced developmental stages than those from the other two groups.
However, there were no significant differences in other traits measured in this study.
Chen et al. (2022) studied the sub-chronic toxicity of TPhP to amphibian tadpoles(Hoplobatrachus rugulosus). GS35 tadpoles were divided in four TPhP-treated groups (three
replicates/group, 30 tadpoles/replicate/30 L): 10, 50, 100, and 200 µg.l-1. After two weeks ofcontinuous TPhP exposure, the dechlorinated tap water culture was switched until the tadpoles
completed metamorphosis (GS46). The endpoints were survival, metamorphosis, and55 (95)

ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC
development. All tadpoles were observed every 24 h until they underwent complete
metamorphosis. The survival and metamorphosis rates were determined within 30 days of subchronic toxicity test, and the metamorphosis time was defined as the interval between the
beginning of TPhP exposure and complete metamorphosis. When the tadpoles completed
metamorphosis, their snout–vent lengths (SVLs) and body masses (BMs) were measured. The
threshold concentration that affected survival and metamorphosis rate was 50 μg/L, while that
affecting the metamorphosis time was 100 µg.l-1. No significant differences were observed in the
body size and condition of the tadpole after metamorphosis; however, BM and TPhP
concentration were negatively correlated.
Conclusion: although indications of effects on thyroid exist, available information are
scarce and possible effects of TPhP on the thyroid pathway were not further
considered in the context of the SVHC identification.
EAS modalities
Based on the available data (Liu et al., 2013b; Li et al., 2019b; Kawashima et al., 2021; He etal., 2021; Zhang et al., 2023), it was considered that TPhP impairs fertility and fecundity in fish.
In Liu et al. (2013b) the effects of TPhP on reproductive endpoints were studied in adult zebrafish(Danio rerio) over 21 days. The study was performed with 12 fish per concentration, exposed to0, 0.04, 0.2 and 1 mg.l-1 nominal TPhP concentrations based on the results of a range-finding
test. No mortality occurred at any concentration. There was a statistically significant decrease
at 0.2 and 1 mg.l-1 of the cumulative egg numbers, the number of spawning events and thehatchability.
Liu et al. (2016) studied the effects of TPhP on zebrafish (Danio rerio) during long-term exposure
covering larval, juvenile and adult life stages. Embryos were collected with 4-h post fertilisationand exposed to 0, 0.005, 0.05 and 0.5 mg.l-1 nominal concentrations of TPhP for 120 days postfertilisation (dpf). There were 3 replicates per treatment, and each replicate contained 100
embryos and 300 ml of test solution. Half of the exposure solution was renewed every 48 h
throughout exposure. Mortality, condition factor (CF), gonodasomatic index (GSI) andhepatosomatic index (HSI) were assessed at 120 dpf. No distinct malformations were observedat any concentration, and the authors concluded that there were no significant differences inmortality between treated groups (although the level of mortality observed, including controls,
was not specified). The CF and GSI were unaffected up to 0.5 mg.l-1 for the larval (14 dpf) andjuvenile (40 dpf) fish. In adult fish (n= 5 per sex), CF (i.e., growth) in male fish was significantly
affected at 0.5 mg.l-1. The gonodasomatic index (GSI) in female zebrafish exposed for 120 days
to TPhP was significantly decreased at 0.005 and 0.5 mg/L with no change in the hepatosomatic
index, suggesting a negative impact on female gonad development.
In Sun et al. (2016), larvae (Japanese medaka) collected in the first hours post-hatching(< 4 hpf) were exposed to TPhP at nominal concentrations of 5, 25, 125, 625 µg.l-1 (no analyticalmeasurement during the test was reported) until 14 d. The media was renewed every 24 h (atleast 90%). Embryos were observed 3 times daily, and the dead larvae were removed. At 60 h,
their heart rates were monitored. At the end of the exposure duration, the body lengths of thelarval medaka were measured. Exposure to TPhP significantly decreased hatchability, delayed
time to hatch, and increased the occurrence of gross abnormalities at the highest exposure
concentration (625 µg.l-1) compared with controls. Moreover, embryo heart rate and body length
were also decreased relatively to the controls in the TPhP exposure groups with concentrations
over 125 µg.l-1.
In Li et al. (2018), larvae (Japanese medaka, male only) collected on the first day post-hatching(0 dph) were exposed to TPhP at nominal concentrations of 1.6, 8, and 40 µg.l-1 (0.134, 0.299
and 1.43 µg.l-1 measured respectively) until 100 dph. A flow-through system with a 2-foldvolume of water flowing through every 24 h was used. After exposure, 16 males were randomly
selected and mated to non-exposed females in clean water for reproductive behaviours and56 (95)

ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC
success tests prior to observation of gonadal intersex. A significant gonadal intersex incidenceof 26% in male medaka was observed at 1.43 µg.l-1 (indicating a potentially feminisation risk
for male fish). In this study chasing trajectory and time, courtship frequency and mating number
were recorded to evaluate the effects of TPhP on reproductive behaviours. After exposure, theincidences of male fish with abnormal chasing behaviour indicated a dose-dependent increase.
Additionally, significant decreases in the fertilisation rate after 1.43 µg.l-1 TPhP exposure and in
the hatching rate after 0.299 and 1.43 µg.l-1 TPhP exposure were observed in the study.
Li et al. (2019b) studied the effects of TPhP on the reproduction of Japanese medaka by an
exposure of 100 days following hatching (FSDT-like test). Medaka (50 individuals, duplicate
experiments) were exposed to TPhP in a continuous flow system at 1.6, 8 and 40 µg.l-1(measured concentrations 1.3, 3.6 and 17.7 µg.l-1). The reproduction test was performed with
6 exposed females paired with control males. Oocyte maturation was quantified by a double
measurement of oocyte size and osp1 gene expression by fluorescence. After 100 days of
exposure, oocytes underwent a delay in maturation characterised by a significant dosedependent decrease in the number of mature and pre-vitellogenic oocytes with more than one
third of females affected. The highest doses induced more substantial effects involving theabsence of oocyte II (stage 3). Cumulative egg production over three days decreased in a dosedependent manner and was significantly affected at the two highest doses (with 39 and 51%
decrease respectively). It should be noted that the concentration of TPhP was higher in the liver
and the ovary than in muscle and brain.
In Kawashima et al. (2021), Japanese medaka (Oryzias latipes) were exposed to TPhP in a flowthrough system for 21 days with 4 replicates per treatment. The age of fish at exposure initiation
was 14 weeks old. Six fish (3 males and 3 females) were used per treatment. The endpoints
measured were mortality, growth (length and weight), fecundity (number of eggs, number of
fertile eggs, fertility rate), GSI, HSI and secondary sexual characteristics. To prevent excessive
lethal effects, the highest test concentration was determined based on acute toxicity on Japanese
medaka. The nominal concentrations were 0 – 20 – 64 – 200 – 640 µg.l-1 (measured
concentration were respectively 0 – 2.13 – 7.19 – 17.1 – 44.9 µg.l-1). Regarding the mortality,
only one female fish died at the highest concentration (44.9 µg.l-1), therefore it is consideredthat the validity criteria was met. The secondary sex characteristics (SSC) were not changed
after exposure to TPhP. A statistically significant decrease in number of total and fertilised eggs
was reported at 44.9 µg.l-1. However, no significant effect on fertility rate (the ratio of thenumber of fertilised eggs to the number of total eggs) was observed.
He et al. (2021) studied acute toxicity with some endocrine indices and effects on reproductionof TPhP on adult zebrafish. The study was conducted with a single dose corresponding to lessthan 10% of the LC50 (based on a range-finding study), i.e., 80 µg.l-1. The test included groupsof 10 separate male or female fish for 21-day exposure. Exposure to TPhP induced a statisticallysignificant increase in the hepatosomatic index (HSI) by a factor of 1.8 and 2.2 for males andfemales, respectively, as well as a decrease in the gonadosomatic index (GSI) in males and anincrease in females (not quantified). These factors were accompanied by histological changes.
The testes and ovaries showed an increase in the early stages of sex cells, and conversely, adecrease in the more developed stages in both sexes indicating an inhibition of gametogenesis(based on qualitative histological observations). Moreover, TPhP significantly reduced the egg
production, the number of spawning, the fertilisation rate and the hatching rate. In conclusion,
the study indicated that TPhP (80 µg.l-1) altered gametogenesis in both sexes of adult zebrafish
resulting in decreased fecundity.
An unpublished study report (2022) aimed to assess the potential endocrine activity and adverse
effects of continuous exposure to TPhP for 73 days i.e., on the early life stages and sexual
differentiation of zebrafish (OECD 234). Thirty fertilised eggs were used for each test with 4
replicates per concentration. Endpoints were determined including hatching success and rates,
mortalities during early life stage and juvenile growth, and maturation phase. Sex ratio wasdetermined macroscopically and by histological examination of the gonads. Measurements weretaken at the end of the study. Additionally, a histopathological examination was performed on
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ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC
the gonads and liver tissues. A maximum tolerated concentration (MTC) of 88.3 µg.l-1 (where noeffect on hatching success, but reduced fish numbers were observed) has been defined by arange-finding study. In the main study, the exposure doses showed a significant deviation (i.e.,
80% or 120%) from the nominal concentrations, and the authors reported the results in mean
measured concentrations of 0; 1.11; 3.01; 7.76; 33.3 and 76.8 µg.l-1. According to the OECD
234 guidelines, all the validity criteria were met.
The survival rates observed were 88%; 84%; 80.0%; 77%; 63% and 51% (control group; 1.11;
3.01; 7.76; 33.3 and 76.8 µg.l-1 respectively). The low survival rate observed at the two highest
doses in the main study was not expected at this level of dose in light of the range finding results.
Mortality was mainly observed during the early life stage of zebrafish until 35 days postfertilisation (dpf), with only few cases of mortality between 35 dpf and test termination (73 dpf).
It should be noted that according to the OECD 234 guideline, if exposure-related mortality
occurs, the number of replicates should be reduced appropriately so that fish density between
treatment levels is kept as equal as possible, as fish density is extremely important for growthand development. Unfortunately, this reduction of replicate was not performed at the two highest
concentrations where mortality occurred. During the early life stage, reduced growth in terms of
total length was observed at the highest tested concentrations. This reduced size was likely due
to the general systemic toxicity of TPhP. However, at 73 dpf, a subsequent exposure to TPhP led
to the increase in mass of the exposed fish in a concentration-dependent manner with astatistically significant difference for the highest dose in males and the two highest
concentrations in females. This mass increase was likely due to reduction of fish density intreatment conditions, resulting from the reduced post-hatch survival rates. The increase in mass
should have been avoided by the reduction of the number of replicates, which was not
performed. The variability of the environmental conditions among the groups could impair theability of the test to show an adverse effect in terms of population development or sex-ratio.
Regarding the apical effect of the OECD 234, namely sex ratio, no significant difference wasobserved, but it should be noted that there was a high proportion of females in the control group(64.8%) compared to exposure groups; even if the acceptance criteria related to proportion ofsex in control group at termination of the test (30% males – 70% females) was fulfilled. The
percentage of females for all the exposed groups increased from 52.6 to 65.4% and thepercentage of males decreased from 46.3 to 33.3% in relation to increasing doses. The numberof undifferentiated intersex was not significantly different (there was only one undifferentiatedfish at the 1.11 and 76.8 µg.l-1 concentrations). It is worth noting that the survival rate might
have altered the statistical power of the study to detect a significant effect on sex ratio in thesegroups. Given the high mortality rate at high doses and the low percentage of males in thosesamples, a deficit of males was observed for these high doses, which might have altered therepresentativeness and therefore the interpretation of the measurements performed, inparticular in males at these doses. It was also not possible to determine whether the mortality
affected one sex more specifically (no sex indication was reported for the dead fish), which may
have interfered with the assessment of sex ratio.
The histopathological analysis of fish gonad revealed that with increasing concentrations, thegonads reached a mature stage more rapidly (for males and females but statistically significant
at the highest concentration for males only). The proportion of stage 2 (the most advanced stage
found) increased from 55 to 90% for testes. Stage 4 in females increased from 15 (control) to
30% (highest dose). The acceleration in gonadal maturation is consistent with the elevatedcirculating steroid levels.
Concerning the histology of female gonads analysed at 3.33 and 76.8 µg.l -1 and in the control
group, a dose-dependent increase in all ovarian pathologies (oocyte atresia, egg debris,
granulomatous inflammation) was observed but without statistical significance in any group.
These pathologies are indicative of the alteration (acceleration) of the oocyte maturation process
and oocyte quality, although not statistically significant.

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ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC
Regarding males, only the testis-ova was measured and was slightly decreased (not statisticallysignificant) with combined acceleration of maturation of the gonad. However, the percentage oftestis-ova males in the control was high (11%), which makes difficult the interpretation of theother doses. Moreover, although testis-ova will likely lead to a decrease in fertility or hatching
success, the design of the test (FSDT, i.e., OECD 234) precludes this type of examination since
FSDT per se does not include the production of a next generation.
In addition to gonad histopathological analysis, liver and heart were examined. Liver from all
test groups was analysed while for the heart, only two groups were examined (control and
76.8 µg.l-1). No statistically significant effect on the severity of the hepatic lesions was observed.
Nevertheless, liver histopathological analysis revealed a dose-dependent decrease in hepatocyte
lipid inclusions in females. In males, a dose-dependent increase in bile duct proliferation andinflammatory foci was observed. The analysis of the liver may reveal a toxic effect or a more
specific effect of lipid metabolism seen in females. Cardiac lesions were also observed in treated
TPhP groups but no statistics could be performed due to the limited number of replicates andtest concentrations.
In Fan et al. (2022) the authors explored the effects of TPhP alone and/or combined to nanotitanium dioxide (nano-TiO2) on the neurodevelopment of zebrafish larvae. Zebrafish embryos(within 2 h after fertilisation) were exposed to concentrations of 0, 8, 24, 72 and 144 µg.l-1 (noanalytical measurement) of TPhP alone for 120h. Zebrafish embryos/larvae were kept in an
incubator at 28 ± 0.5 ◦C, with a light-dark ratio of 14 h/10 h. During the period of exposure, thesolutions were renewed daily to keep the concentrations constant. No developmental toxicity
was detected by treating with TPhP at 8 and 24 µg.l-1, while higher concentrations of TPhP
(72 µg.l-1 and 144 µg.l-1) significantly altered the hatching rate, the heart rate, the malformation
rate, and the body length in zebrafish larvae (only at 144 µg.l-1). Note that at the highest dosethe inhibitions on the hatching rate, heartbeat and body length in zebrafish larvae weresignificantly promoted in the co-exposure groups where TiO2 was added, compared to TPhP
alone.
In Zhang et al. (2023) the authors investigated the effects of TPhP exposure on developmental
parameters, locomotor behaviour, oxidative stress, apoptosis and transcriptional levels inzebrafish at different developmental stages. Zebrafish embryos (0 – 2 hpf) were exposed toconcentrations of 0, 20, 50, 100, 500 and 1000 µg.l-1 of TPhP for 7 days (based on a range
finding study with a 96h LC50 value of TPhP on zebrafish embryos of 5.13 mg.l-1). The results
showed that the survival rate decreased significantly compared to the control at theconcentration of 500 µg/L from 120 hpf. In the same way, the hatching rate decreased at 50µg/L
at 48 hpf and 500 µg/L at 72 hpf compared to the control. TPhP also significantly increased theheart rate of zebrafish larvae at 48 hpf (100 and 1000 µg.l-1) and at 60 hpf (500 and 100 µg.l1) but significantly decreased at 96 hpf (500 µg.l-1). Microscopic observations also showed thatthe exposure of TPhP led to several malformation symptoms such as yolk sac edema, spinal
curvature, abnormal body pigmentation, pericardial edema, small eyes, etc. Concerning the
behaviour changes, TPhP could affect the embryonic spontaneous movement. Regarding the
locomotor behaviour and the neurotoxicity, all TPhP treated groups caused statistically significant
increase in swimming distance and movement trajectory of zebrafish larvae except for the 50µg.l-1 groups. The authors also found that TPhP increased the number of apoptotic cells in thehead, heart and intestine of zebrafish. In the head region, the number of apoptotic cells werelocalised in the eyeball of zebrafish larvae.
Conclusion: in vivo studies on fish reproduction show an alteration of gametogenesis
in both sexes with a disruption of testis and ovary maturation followed by a decrease
in reproductive success in terms of fecundity (decrease in egg production, spawning
events) and fertility (impaired hatchability and fertilisation rate). In vivo studies onamphibian development show that TPhP could affect tadpole survival andmetamorphosis.

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ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC
Analysis of systemic toxicity in relation to the observation of EAS-related adverse
effects
As recommended in the EDC guidance (ECHA/EFSA, 2018), adverse effects that are non-specificsecondary consequences of other toxic effects shall not be considered for the identification of
the substance as endocrine disruptor. The top concentration selected for the conduction of theecotoxicological studies should provide information on substance toxicity at an exposure of thetested agent that should be tolerated without inducing significant chronic physiologicaldysfunctions, be compatible with animal survival and permits data interpretation in the context
of the use of the study. It is recommended that a Maximum Tolerated Concentration (MTC)should be considered for the evaluation of changes which could be due to excessive systemic
toxicity. In ecotoxicology, the MTC is defined as the highest test concentration of the chemical
which results in less than 10% mortality.
For each study, information on mortality when available has been reported in the Table above.
Data do not systematically allow to determine a MTC value.
Japanese medaka (Oryzias latipes)
Four studies (Sun et al., 2016 - Li et al., 2018 - Li et al., 2019b - Kawashima et al., 2021) ofinterest are available. The only study to provide information on the survival rate of fish after
exposure of TPhP is Kawashima et al., 2021.
Table 13: Summary table of survival data - Japanese medaka (Oryzias latipes)
Ref.

Kawashima
et al. 2021

Age
study
start

at

16±2
weeks old

Durationofexposure

21d

Media renewal

Flow-through(5 times/day)

Nominalconc. (µg.l-1)

Measuredconc. (µg.l-1)

Control
group
20
64
200
640

Control
group
2.13
7.19
17.1
44.9

Survival rate

0%

0%
0%
0%
95.8% (1 female)

It is therefore not possible to determine a lowest observed effect concentration (LOEC) on
mortality from this study. However, MTC was not exceeded. It is therefore not possible todetermine a systemic toxicity threshold from survival rate for Japanese medaka (Oryzias latipes)
from these studies.

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ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC
Zebrafish (Danio rerio)
Eleven studies (Liu et al., 2012 - Liu et al., 2013a - Liu et al., 2013b - Kim et al., 2015 - Liu etal., 2016 - Liu et al., 2019- He et al., 2021 - Lee et al., 2022 - Unpublished study report, 2022
– Fan et al., 2022 - Zhang et al., 2023) of interest are available and information on mortality issummarised below.
Table 14: Summary table of survival data - Zebrafish (Danio rerio)
Ref.

Age
study
start

Liu et al.,
2012

at

4 months

Durationofexposure

14d

Media
renewalEvery otherday

Nominalconc.
(mg.l-1)
Control group
0.04
0.2

Measuredconc. (mg.l-1)

NAM

1

Survival rate

No significant
mortality (novalue provided)

No value for survival rate. MTC was not exceeded
Control group
Liu et al.,
2013b

4-5
months21d

0.04<LOD (48h)0.2<LOD (48h)10.38 (48h)Every 48h

No mortality (novalue provided)

No mortality at 0.38 mg.l-1. MTC was not exceeded
Control group
Liu et al.,
2019

4-5
months14d

at least
three times
per week

0.04
0.2
1

0.03 (0h)<LOD (48h)0.15 (0h)<LOD (48h)0.87 (0h)0.42 (48h)No mortality (novalue provided)

No mortality at 0.42 mg.l-1. MTC was not exceeded
He et al.,
Adult
21d
Every day
0.08
NAM
2021
No information.
Impossible to determine a systemic toxicity threshold from survival rate

No information

Control group0.02
0 (120hpf)
> 90%
4hpf
120hpfat 48h only
0.2
0 (120hpf)> 90%
21.39 (120hpf)> 90%
Mortality < 10% at 1.39 mg.l-1 . MTC was not exceeded.
Control group
91.8%
0.04
92.5%
Kim et al.,
3hpf
7d
50% daily
2015
0.2
NAM
92%
0.5
88.7%
Mortality > 10% at 0.5 mg.l-1 but not > 10% compared to mortality in the control group. MTC is notconsidered to have been exceeded.
Control group0.0008 (24h)0.005< LOD (48h)Liu et al.,50% every
No significant
4hpf
120d
2016
48h
0.0055 (24h)mortality (no0.05< LOD (48h)value provided)
0.5
0.131 (24h)
Liu et al.,2013a

61 (95)

ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC
0.011 (48h)Impossible to determine a correct range of exposure concentrations. No value for survival rate.
Impossible to determine a systemic toxicity threshold from survival rate. MTC was not exceeded.
Control group0.32
No significant
Lee et al.,
No
72hpf
24h
0.97
mortality (no2022
information
NAM
value provided)
3.2
9.8
< 50%
Low survival rate at 9.8 mg.l-1. No value for survival rate. MTC may have been exceeded.
Control group
88%
0.001
0.001
84%
Unpublished
Flow0.0032
0.003
80%
study
<12hpf60d
throughreport,
0.01
0.008
77%
system
2022
0.032
0.033
63%
0.1
0.077
51%
Mortality in the control group > 10%. Mortality > 10% compared to mortality in the control group
above 0.008 mg/l. 0bservation of mortality in controls demonstrate that TPhP exposure is not the only
driver for mortality.
Control group
87%
0.008
86%
Fan et al.,
2hpf
120h
daily
0.024
84%
2022
NAM
0.072
82%
0.144
81%
Mortality in the control group > 10% but not > 10% compared to mortality in the control group.
Observation of mortality in controls demonstrate that TPhP exposure is not the only driver for
mortality. MTC is not considered to have been exceeded.
Control group
>80%
0.02
NAM
90%
0.05
NAM
90%
Zhang et al.
2hpf
7d
90% daily
2023
0.1
NAM
90%
0.5
NAM
30%
1
NAM
20%
No exact value for the survival rate (observations from a graph).
Mortality in the control group > 10%. Mortality > 10% compared to mortality in the control group
above 0.5 mg/l. Observation of mortality in controls demonstrate that TPhP exposure is not the only
driver for mortality.

NAM: no analytical measurement
Data do not systematically allow to determine a MTC value. It is noted that due to limitations inthe studies (absence of measured concentrations) and differences in experimental design (stage
and duration of exposure), it is impossible to determine a systemic toxicity threshold fromsurvival rate for Zebrafish (Danio rerio).
Overall, mortality and its possible interference with EAS-adverse effects shall be interpreted ineach study independently.

Conclusion regarding ED properties relevant for environment
Adverse effects relevant for ED identification
The available data indicate that exposure to TPhP damages fish reproduction by impacting
processes and events during gametogenesis (oocyte growth, ovary and testis maturation),
disrupting fish fecundity (eggs production and spawning) and fertility (fertilisation and hatching).
Changes in fecundity and fertility are apical effects, which inform about potential adverse effects
at fish population level.

62 (95)

ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC
The decrease in mature spermatocytes (He et al., 2021) suggests a potential delay inspermatogenesis. A significant gonadal intersex incidence of 26% in male medaka was observed
at 1.43 µg.l-1 (Li et al., 2018), indicating a potential feminisation risk for male fish. A disruption(dose-dependent increase) of male-typical reproductive behaviours before spawning (chasing
trajectory and time, courtship frequency and mating number) observed by Li et al. (2018) also
supports the reproduction impairment of male fish after exposure to TPhP. This effect can explain
the observed decrease in fertilisation rate.
Data show an inhibition of the ovarian development in female fish (He et al., 2021) andalterations of the ovaries (Li et al., 2019b; He et al., 2021). These alterations and development
retardation can affect the fecundity of female fish with the observed decrease in egg production
and spawning.
Despite the observation of some effects in the FSDT study, it should be noted that the quality ofthis study did not allow to evaluate properly EAS mediated parameters.
The decrease in egg production ( Liu et al., 2013b; Li et al., 2019b, He et al., 2021; Kawashima
et al., 2021) is a clear effect indicator of the impaired reproductive ability, reinforced by adecrease of spawning events (Liu et al., 2013b; He et al., 2021), an insufficient fertilisation rate
(Li et al., 2018; He et al., 2021) and a decrease in hatchability (Liu et al., 2013b; Li et al., 2018;
He et al., 2021), disrupting the generation of new individuals. This impact on fish reproduction
could affect fish population stability and is considered as an adverse effect relevant at populationlevel.
Moreover, it is important to note that several adverse effects on altered reproductive capacityof fish are observed in several studies at environmentally relevant levels without concurrent
systemic effects. Indeed, no specific mention of mortality was reported in several studies (He etal., 2021; Li et al., 2018; Li et al., 2019b) although the absence of specific information on thisparameter leaves some uncertainties. Most importantly, effects on hatching rate, fecundity andfertility were clearly observed without any concurrent mortality in Liu et al. (2013b) and
Kawashima et al. (2021).
TPhP shows adverse effects on fertility and fecundity of fish at environmentally
relevant levels. The impact on fish reproduction could affect fish population stability
and is considered as an adverse effect relevant at population level. These effects
cannot be considered as a secondary non-specific consequence of general toxicity.
Data (in vivo) providing scientific evidence of an adverse effect of TPhP on the reproductive
system are summarised in Table 13 below.

63 (95)

Table 15: Line of evidence in relation to TPhP sexual dysfunction in in-vivo studies
Endpoint

Biologicalmodel

Species

Exposure
duration

Adult orjuvenile(when
exposed)Lowestsignificant
concentration

Observed effects

Reference

Hatching rate-

Zebrafish

21 daysAdult

0.2 mg.l-1↓

Liu et al., 2013b

Hatching rate-

Zebrafish

21 daysAdult80 µg.l-1↓#

He et al., 2021

Hatching rate-

100 days

Juvenile

0.299 µg.l-1↓#

Li et al., 2018
Li et al., 2018

JapaneseMedakaJapaneseMedaka100 days

Juvenile

1.43 µg.l-1↑ intersex incidence inmale fish

Zebrafish

73 daysJuvenile-##-

JapaneseMedaka100 days

Juvenile

1.43 µg.l-1

Fecundity-

Zebrafish

21 daysAdult

0.2 mg.l-1

Fecundity-

JapaneseMedaka100 days

Female juvenile

0.36 µg.l-1#↓ egg production

Li et al., 2019b

Fecundity/fertility-

Zebrafish

21 days

Adult-

Fertility rate: no change

Liu et al., 2013b

Fecundity/fertility-

JapaneseMedaka100 days

Juvenile

1.43 µg.l-1↓# fertilisation rate

Li et al., 2018

Sex ratio-

Sex ratio-

Reproductive behaviour#

No change

Abnormal chasing
behaviour in male fish
↓ egg production, spawningevent#↓ fertilisation rate,
cumulative eggs, spawningevent↓ egg production andfertilised eggs*↑## testis maturation
stages↓# number of mature
oocytesUnpublished study
report, 2022
Li et al., 2018
Liu et al., 2013b#

Fecundity/fertility-

Zebrafish

21 daysAdult80 µg.l-1

Fecundity/fertility-

JapaneseMedaka21 daysAdult

44.9 µg.l-1

Histology

TestesZebrafish

73 days

Male juvenile

76.8 µg.l-1

Histology

Ovaries

JapaneseMedaka100 days

Female juvenile

0.13 µg.l-1

Histology

OvariesZebrafish

21 daysFemale adult80 µg.l-1↓# ovary maturation stages

He et al., 2021

Histology

TestesZebrafish

21 days

Male adult80 µg.l-1↓# testis maturation stages

He et al., 2021*: however no significant effect on fertility rate (ratio)#
No information on mortality##
Mortality > 10% observed, with mortality > 10% also in the control group
MTC not exceeded for the other studies

He et al., 2021
Kawashima et al., 2021
Unpublished study
report, 2022
Li et al., 2019b

Endocrine activity
In vitro and in vivo studies are available supporting an estrogenic activity of TPhP, assummarised in Table 14 below.
Molecular Initiating events (MIEs)-

Nuclear estrogen receptors

The estrogenic activity is mainly mediated via nuclear estrogen receptors (nERs), which function
as transcription factors upon ligand binding for genomic responses. In most vertebrates,
including mammals, birds, and some lower vertebrates, two estrogen receptors exist: thenuclear intracellular receptors ER and ER, considered to mediate the effect of E2 and VTG inliver of most fish species. Data show that TPhP has an agonist activity on human ER and ERβ
receptors and decrease the binding affinity of ER (Liu et al., 2012; Kojima et al., 2013; Zhang
et al., 2014; Kojima et al., 2016; Medvedev et al., 2020; Ji et al., 2022). The agonist activity isalso reported for the medaka ER1 (Kawashima et al., 2021) and for the fish ER2β, frog ER1,
turtle ER1 and chicken ER1 (Medvedev et al., 2020).
In some yeast two-hybrid assays with human and medaka ERα, an antagonist effect is shown
indicating a competition between TPhP and E2 for binding to ERα (Ji et al., 2020; Li et al., 2018).
Zhang et al. (2014) demonstrated a binding affinity of TPhP to hERα with a docking approach.
Moreover, TPhP can induce the ERE pathway demonstrated by a dose-response of the agonist
effect in MVLN cells (transfected with the human ERE-luciferase reporter gene) (Ji et al., 2020).
This estrogenic activity is supported by proliferation assays on MCF-7 cells that show TPhP
promotes cell proliferation (Zhang et al., 2014; Krivoshiev et al., 2016; Ji et al., 2020).
-

GPER (membrane estrogen receptor)

The estrogenic activity can also be mediated via membrane estrogen receptors as G proteincoupled membrane receptors (GPER). The role of GPER in oocyte maturation is well defined inzebrafish (Thomas, 2017; Chen et al., 2019).
In Guan et al. (2022), the authors report that TPhP binds GPER with an IC50 of 4.2 µM. This isconfirmed by E2-F displacement experiments. Combined transcriptome and proteome analysis
show that TPhP can exert its estrogen-like proliferation-promoting effect through the interactionof the PI3K-Akt signalling pathway, MAPK signalling pathway, and EGFR signalling pathway,
which are downstream pathways mediated by GPER. In Ji et al. (2022), the assay on SBRK3cells shows that TPhP can interact with GPER and activate cAMP formation, which is one of therapid signalling pathways induced by GPER activation. However, this activation is 10-times lessefficient than the reaction induced by E2. Nevertheless, this activation via GPER is also confirmedby the results obtained with a TPhP co-exposure with the specific GPER30 inhibitor, G15, which
inhibits the effects of TPhP in the assay.
-

Aromatase activity

Aromatase (CYP19) catalyses the conversion of androgenic steroids to estrogenic steroids(Conley and Hinshelwood, 2001). The CYP19 plays a role in signaling pathways critical for sexual
development and fertility. TPhP induced an increase of the expression of the CYP19 gene (Liu etal., 2012; Liu et al., 2013b; Liu et al. 2016; Ji et al., 2022) and 17β-HSD1 gene (Liu et al.,2013b; Liu et al., 2016; Ji et al., 2022), which is linked to the steroidogenic pathway. In Ji et al.
(2022), a decrease of estrone was observed while CYP19 is upregulated and E2 level increased.
The reason given by the authors was that the conversion activity of estrone to E2 by 17β-HSD1
was stronger than the activity of CYP19 to estrone synthesis. TPhP affected steroidogenesis byincreasing the estrogen levels (17ß-estradiol) via the up-regulation of CYP19.

ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC
Steroid hormone level and Vitellogenin
During medium-term exposure, TPhP induces an increase in circulating E2 concentrations inzebrafish and medaka (Liu et al., 2012; Liu et al., 2013b; Liu et al., 2016, Li et al., 2018; Li etal., 2019b, Unpublished study report, 2022) and a decrease in 11-KT (Liu et al., 2012; Liu etal., 2013b; Liu et al., 2016; Li et al., 2018; Li et al., 2019b). TPhP also induces an increase of
VTG concentrations in Liu et al. (2013b) and Unpublished study report (2022), which support
the estrogenic activity of TPhP.
Nevertheless, in one study (Li et al., 2019b) carried out on Japanese medaka, a decrease of E2
level was noted after 100-day exposure of female larvae (age of fish at exposure initiation),while an increase of E2 was observed after 21-day exposure of adult females (age of fish at
exposure initiation).
The observations of VTG concentrations suggested an estrogenic activity of TPhP in female andmale zebrafish, with increased plasmatic concentration of VTG (Liu et al., 2013b) andUnpublished study report (2022). Some results were also reported with decreased or unchanged
concentration in plasma VTG of female medaka (Kawashima et al., 2021) or in whole-body
homogenates of male and female zebrafish (He et al., 2021). Vtg1 gene expression in liver wasincreased in male zebrafish in one assay (Liu et al., 2012), while the expression in liver of vtg1
and 2 were decreased in other assays (Liu et al., 2012 (for females); Li et al., 2019b). These
different perturbations of circulating steroid concentrations relate to the fish developmental
stage, species and tested concentrations.
Data therefore provide in vitro and in vivo evidence that TPhP has EAS activity. In
absence of mortality reported in several studies, this activity cannot be considered asa secondary non-specific consequence of general toxicity.

66 (95)

Table 16: Line of evidence in relation to TPhP EAS activity
Assay category

Species/
Endpoint

Biological model

Exposure
time

Adult orJuvenile(when
exposed)Parameter

Lowestsignificant
concentration

Observed effects

Reference

In vitro endocrine activity
TransactivationhERα

CHO-K1 cells

24h

-

REC20

-

Agonist: 0.27 µM

Zhang et al., 2014

TransactivationhERαYeast cells

-

-

REC20

-

Agonist: 0.65 µM

Zhang et al., 2014

TransactivationhERα

MCF-7 cells

60h

-

REC20

-

Agonist: 0.1 µM

Zhang et al., 2014

TransactivationhERα

CHO-K1 cells

24h

-

REC20

-

Weak agonist: 4.9µM

Kojima et al., 2013

TransactivationhERα

CHO-K1 cells

24h

-

REC20

-

Weak agonist: 4.6µM

Kojima et al., 2016

Transactivation

hERß

CHO-K1 cells

24h

-

REC20

-

Weak agonist: 6.5µM

Kojima et al., 2013

Transactivation

hERß

CHO-K1 cells

24h

-

REC20

-

Weak agonist: 7.3µM

Kojima et al., 2016

Hormonemeasurement

Estradiol level

H295R cells

48h

Adult

b

1 mg.l-1↑

Liu et al., 2012

Hormonemeasurement

E2 level

H295R cells

48h

-

b

1 µM

↑

Ji et al., 2022

Hormonemeasurement

Testosteronelevel

H295R cells

48h

Adult

b

1 mg.l-1↑

Liu et al., 2012

Hormonemeasurement

Testosteronelevel

H295R cells

48h

-

b

1 µM

↓

Ji et al., 2022

Hormonemeasurement

E2/T ratio level

H295R cells

48h

Adult

b

0.1 mg.l-1↑

Liu et al., 2012

Hormonemeasurement

E2/T ratio

H295R cells

48h

-

b

1 µM

↑

Ji et al., 2022

Gene expression

CYP19A1

H295R cells

48h

Adult

b

1 mg.l-1↑

Liu et al., 2012

ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHCTransactivation

ER

MCF-7 cells (MVLN
cells)

72h

Adult

b

0.001 mg.l-1

TransactivationmERαMVLN cell

24h

-

b

IC50

Transactivation

ER

MCF-7 cells (MVLN
cells)

72h

-

EC50

EC50

Binding-

SBRK3 cells

24h

-

b

-

Gene expression

Cyp19

H295R cells

48h

-

b

1 µM

↑

Ji et al., 2022

Transactivation

mEsr1

-

-

-

b

-

Agonist activity

Kawashima et al.,2021

Antagonism
Antagonist activity(IC 50 = 29 µM)
Agonist activity(EC50 = 1.2 x 10-5
M)Interaction withGPER via cAMP
formation

Liu et al., 2012
Li et al., 2018
Ji et al., 2022
Ji et al., 2022

In vivo endocrine activity
Hormonemeasurement

E2 level

Zebrafish plasma

120 daysMaleembryob

5 µg.l-1↑

Liu et al., 2016

Hormonemeasurement

E2 level

Medaka plasma

100 daysMaleembryob0.299 µg.l-1↑#

Li et al., 2018

Hormonemeasurement

E2 level

Zebrafish plasma

14 days

Male adultb

1 mg.l-1↑

Liu et al., 2012

Hormonemeasurement

E2 level

Zebrafish plasma

21 days

Male adultb

0.2 mg.l-1↑

Liu et al., 2013b

Hormonemeasurement

E2 level

Zebrafish plasma

14 daysFemaleadultb

1 mg.l-1↑

Liu et al., 2012

Hormonemeasurement

E2 level

Zebrafish plasma

21 daysFemaleadultb

1 mg.l-1↑

Liu et al., 2013b

Hormonemeasurement

E2 level

Medaka plasma

21 daysFemaleadultb1.72 µg.l-1↑#

Li et al., 2019b

Hormonemeasurement

E2 level

Zebrafish plasma

73 daysFemaleembryob3.01 µg.l-1↑##

Unpublished study
report, 2022

Hormonemeasurement

E2 level

Zebrafish plasma

120 days

Femaleembryob

5 µg.l-1↑

Liu et al., 2016

Hormonemeasurement

E2 level

Medaka plasma

100 days

Femaleembryob

1.77 µg.l-1↓#

Li et al., 2019b68 (95)

ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC
Hormonemeasurement

E2 level

Zebrafish plasma

21 days

Male adultb-

No change

He et al., 2021

Hormonemeasurement

E2 level

Zebrafish plasma

21 daysFemale
adult

b

-

#

No change

He et al., 2021

Hormonemeasurement

T levelZebrafish plasma

14 days

Male adultb

1 mg.l-1↓

Liu et al., 2012

Hormonemeasurement

T levelZebrafish plasma

14 daysFemaleadultb-

No change

Liu et al., 2012

Hormonemeasurement

T levelZebrafish plasma

21 daysFemaleadultb

1 mg.l-1↓

Liu et al., 2013b

Hormonemeasurement

T levelMedaka plasma

100 daysMaleembryob1.43 µg.l-1↓#

Li et al., 2018

Hormonemeasurement

T levelMedaka plasma

21 daysFemaleadultb1.72 µg.l-1↑#

Li et al., 2019b

Hormonemeasurement

T levelMedaka plasma

100 days

Femaleembryob0.13 µg.l-1↓#

Li et al., 2019b

Hormonemeasurement

T levelZebrafish plasma

21 days

Male adult

b

-

#

No change

He et al., 2021

Hormonemeasurement

T levelZebrafish plasma

21 daysFemale
adult

b

-

#

No change

He et al., 2021

Hormonemeasurement

E2/T level

Zebrafish plasma

21 daysFemaleadultb

0.2 mg.l-1↑

Liu et al., 2013b

Hormonemeasurement

E2/T level

Zebrafish plasma

21 daysFemaleadultb

80 µg.l-1↓#

He et al., 2021

Hormonemeasurement

E2/T level

Zebrafish plasma

21 days

Male adult

b

-

#

No change

He et al., 2021

Hormonemeasurement

E2/T level

Zebrafish plasma

21 days

Male adultb0.04 mg.l-1↓

Liu et al., 2013b

Hormonemeasurement

E2/T level

Zebrafish plasma

14 days

Male adultb

1 mg.l-1↑

Liu et al., 2012

Hormonemeasurement

E2/T level

Zebrafish plasma

14 daysFemaleadultb-

No change

Liu et al., 201269 (95)

ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC
Hormonemeasurement

11-KT level

Zebrafish plasma

14 daysFemaleadultb-

No change

Liu et al., 2012

Hormonemeasurement

11-KT level

Zebrafish plasma

120 days

Femaleembryob

500 µg.l-1↓

Liu et al., 2016

Hormonemeasurement

11-KT level

Medaka plasma

100 days

Femaleembryob0.13 µg.l-1↓#

Li et al., 2019b

Hormonemeasurement

11-KT level

Zebrafish plasma

120 daysMaleembryob

5 µg.l-1↓

Liu et al., 2016

Hormonemeasurement

11-KT level

Medaka plasma

100 daysMaleembryob1.43 µg.l-1↓#

Li et al., 2018

Hormonemeasurement

11-KT level

Zebrafish plasma

73 daysMaleembryob3.01 µg.l-1↑##

Unpublished study
report, 2022

Hormonemeasurement

11-KT level

Zebrafish plasma

21 daysFemaleadultb

1 mg.l-1↓

Liu et al., 2013b

Hormonemeasurement

11-KT level

Zebrafish plasma

14 days

Male adultb

1 mg.l-1↓

Liu et al., 2012

Hormonemeasurement

E2/11-KT level

Zebrafish plasma

120 days

Femaleembryob

500 µg.l-1↑

Liu et al., 2016

Hormonemeasurement

E2/11-KT level

Zebrafish plasma

120 daysMaleembryob

5 µg.l-1↑

Liu et al., 2016

Hormonemeasurement

E2/11-KT level

Zebrafish plasma

14 daysFemaleadultb

1 mg.l-1↑

Liu et al., 2012

Hormonemeasurement

E2/11-KT level

Zebrafish plasma

21 daysFemaleadultb

1 mg.l-1↑

Liu et al., 2013b

Hormonemeasurement

E2/11-KT level

Zebrafish plasma

14 days

Male adultb

1 mg.l-1↑

Liu et al., 2012

Hormonemeasurement

E2/11-KT level

Zebrafish plasma

21 days

Male adultb

0.2 mg.l-1↑

Liu et al., 2013b

Hormonemeasurement

VTG level

Zebrafish plasma

73 daysFemaleembryob33.3 µg.l-1↑##

Unpublished study
report, 2022

Hormonemeasurement

VTG level

Zebrafish plasma

73 daysMaleembryob33.3 µg.l-1↑##

Unpublished study
report, 2022

70 (95)

ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC
Hormonemeasurement

VTG level

Zebrafish plasma

21 daysFemaleadultb

0.2 mg.l-1↑

Liu et al., 2013b

Hormonemeasurement

VTG level

Zebrafish plasma

21 daysFemaleadultb

80 µg.l-1↓#

He et al., 2021

Hormonemeasurement

VTG level

Zebrafish plasma

21 days

Male adult

b

-

#

No change

He et al., 2021

Hormonemeasurement

VTG level

Japanese Medaka
plasma21 daysFemaleadultb

7.19 µg.l-1↓

Kawashima et al.,2021

Hormonemeasurement

VTG level

Zebrafish plasma

21 days

Male adultb

1 mg.l-1↑

Liu et al., 2013b

Gene expression

vtg1

Medaka liver

100 days

Femaleembryob0.13 µg.l-1↓#

Li et al., 2019b

Gene expression

vtg1

Zebrafish plasma

14 daysFemaleadultb

1 mg.l-1↓

Liu et al., 2012

Gene expression

vtg1

Zebrafish plasma

14 days

Male adultb0.04 mg.l-1↑

Liu et al., 2012

Gene expression

vtg2

Medaka liver

100 days

Femaleembryob0.36 µg.l-1↓#

Li et al., 2019b

Gene expression

vtg2

Medaka liver

21 daysFemaleadultb1.72 µg.l-1↓#

Li et al., 2019b

Gene expression

cyp19a mRNA

Zebrafish plasma

14 daysFemaleadultb

1 mg.l-1↑

Liu et al., 2012

Gene expression

cyp19a mRNA

Zebrafish plasma

14 days

Male adultb

1 mg.l-1↑

Liu et al., 2012

Gene expression

ER2b

Zebrafish

5 daysEmbryosb

2 mg.l-1↑

Liu et al., 2013a

Gene expression

CYP19A

Zebrafish – ovaries

21 daysFemaleadultb

1 mg.l-1↑

Liu et al., 2013b

CYP19b

Zebrafish – brain

21 daysFemaleadultb

0.2 mg.l-1↑

Liu et al., 2013b

ERα

Zebrafish – brain

21 daysFemaleadultb

1 mg.l-1↑

Liu et al., 2013b

ER2β1

Zebrafish – brain

21 daysFemaleadultb

1 mg.l-1↑

Liu et al., 2013b

CYP19A

Zebrafish – testis

21 days

Male adultb

1 mg.l-1↑

Liu et al., 2013b

Gene expression
Gene expression
Gene expression
Gene expression

71 (95)

ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC
Gene expression

CYP19B

Zebrafish – brain

21 days

Male adultb

1 mg.l-1↑

Liu et al., 2013b

Gene expression

ERα

Zebrafish – brain

21 days

Male adultb

1 mg.l-1↑

Liu et al., 2013b

Gene expression

ER2β1

Zebrafish – brain

21 days

Male adultb

0.2 mg.l-1↑

Liu et al., 2013b

Gene expression

ERα

Zebrafish – brain

120 days

Femaleembryob

500 µg.l-1↑

Liu et al., 2016

Gene expression

CYP19A

Zebrafish – ovaries

120 days

Femaleembryob

500 µg.l-1↑

Liu et al., 2016b: qualitative assessment only, no parameter calculated.
#
No information on mortality##
Mortality > 10% observed, with mortality > 10% also in the control group
MTC not exceeded for the other studies

72 (95)

Plausible link between adverse effects and endocrine activity
The available data indicate that an exposure of TPhP damages fish reproduction by impactingsome processes and events during gametogenesis, disrupting fish fecundity (egg production andspawning) and fertility (hatching and fertilisation). Changes in fecundity and fertility are apicaleffects, which inform about potential adverse effects at the population level.
Although these reproduction parameters are sensitive to substances interfering with the sex
hormone system, they are not considered as “EATS-mediated” as they might be influenced by
non-endocrine factors such as systemic toxicity. Nevertheless, they can be used in a weight ofevidence approach to draw a conclusion on a specific endocrine pathway.
These adverse effects are related to the disturbances in steroid synthesis and gametogenesis.
In fact, all available in vitro and in vivo studies demonstrate that TPhP exerts an action on theendocrine hormone balance in fish. Depending on the developmental stage, species andconcentration, antagonist and agonist effects on nuclear and membrane estrogen receptors areobserved in organisms as well as on transcription of genes involved in steroidogenesis, leadingin vivo to perturbations of circulating steroid concentrations. Consistency between endocrine
perturbation and adverse effect on reproduction is generally observed in the available studies.
The data were analysed separately for males and females as the major steroid hormones in theregulation of fish gametogenesis is the estrogen E2 in females and the androgen 11KT in males.
Reproductive dysfunction in male fish
Based on the available information for males and lines of evidence for adversity and endocrine
activity, the following potential sequence of causality linked events at different levels of biologicalorganisation, that potentially lead to an adverse ecotoxicological effect (reproductive dysfunction
in fish), is represented in the following figure.
Adverse
Outcome
(AO)

KEs

MIEs

Increase of VTG levels inplasmaGPER
activation
and/or ER
activation
Alteredreproduction

Aromataseactivity
increased

Increase of E2
levels andE2/11KT in
plasmaChanges in gonad
histopathology and /orphysiology

Figure 4: Potential sequences of linked events at different levels of biological organisation that
potentially lead to an adverse ecotoxicological effect (reproductive dysfunction in male fish)73

ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC
The decreased amount of mature spermatocyte cells (He et al., 2021) suggests a potential delayof spermatogenesis following the exposure to TPhP. A disruption (dose-dependent increase) of
male-typical reproductive behaviours before spawning (chasing trajectory and time, courtshipfrequency and mating number) observed in Li et al. (2018) also supports the reproduction
impairment of male fish by exposure to TPhP.
CYP19A and CYP19B were significantly upregulated in the testis and the brain respectively (Liuet al., 2012; 2013 b) and could increase conversion of T to E2. Moreover, estrogen receptors(ERα, ER2β1) were up-regulated in the brain (Liu et al., 2013b).
Generally, after TPhP exposure of male fish, plasma E2 increased significantly, plasma 11-KT
decreased and E2/11-KT ratio increased (Liu et al., 2012; Liu et al., 2016; Li et al., 2018; Liu etal., 2013b). However, this trend could not be verified in the unpublished study report (2022),
as E2 levels were not measured in males and consequently the E2/11KT ratio could not becalculated. 11-KT plays an important role in the spermatogonial proliferation. A decreased 11KT level could stop meiosis and germ cell maturation (Schulz et al., 2010). The decrease of 11KT and the increase in E2 concentrations in plasma revealed by E2/11KT ratio (indicative of
estrogenicity) can be associated with reduced semen production and density in male fish. Finally,
the increase of the expression in liver of VTG1 gene (Liu et al., 2012) and the increase of plasma
VTG levels in male fish exposed to TPhP (Liu et al., 2013b; Unpublished study report, 2022) areconsistent with the observed changes in sex hormones (increased E2 synthesis). These events
support the reproduction impairment of male fish observed after exposure to TPhP. The following
table highlights these relationships. It has to be noted that the reported concentrations are thosefor which the measured parameters are significant.
Table 17: Dose-response and temporal concordance between the key events for male fish
[Zebrafish – Japanese Medaka - male] potential dose–response and temporal concordance between the
key events and AO
Observed
Reference
Dose
Estradiol
11-KT
E2/11KT
Testis histopathology
VTG level
Adverse-1(µg.l )
level
level
ratio
(abnormalities)
effect0.299#

1.43#↑ (100days)↓ hatchingrate

Li et al., 2018

↑ (100days)↑ intersex
incidence,
abnormalchasingbehaviour↓ fertilisationrate.

Li et al., 2018

Unpublishedstudy report,
2022

↑ (73
days)*

3.01##
5↓ (100days)↑ (120days)↓ (120days)

7.76##↑(’73days)*

33.3##↑ (73days)*↑ (120days)

Liu et al., 2016
Unpublishedstudy report,
2022
Unpublishedstudy report,
2022

↑ (73
days)
↑ (14
days)***

40

Liu et al., 2012
↓ Fertilised
eggs**

44.9
50↓ (120days)

76.8##↑ (73days)*↑ (120days)

Kawashima etal., 2021
Liu et al., 2016

↑ testis maturation
stage (73 days)

Unpublishedstudy report,2022

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ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC

80

↑ immature
spermatocytes(spermatogonia andspermatocyte)↓ mature
spermatocytes(spermatids andspermatozoa)

#

↑ (14
days)***

200
200↓ hatchingrate↓ fertilisation
rate

↑ (21
days

500

↓ (120
days)
↓ (14
days)

↑ (21
days)
↑ (120
days)
↑ (14
days)

He et al., 2021

Liu et al., 2012
↓ hatchingrate

Liu et al.,2013b
Liu et al., 2016

↑ (14
Liu et al., 2012days)***↑ (21Liu et al.,
1000
days)
2013b
* Unpublished study report (2022), where E2 levels were not measured in males and consequently theE2/11KT ratio could not be calculated.
** However no significant effect on fertility rate (ratio).
*** Transcriptional gene VTG1.
#
No information on mortality##
Mortality > 10% observed, with mortality > 10% also in the control group
MTC not exceeded for the other studies
Blank cells: parameter not investigated at this concentration.
1000

↑ (14
days)

Dose–response and temporal concordance between the key events and AO are often observedat concentration where systemic toxicity was not observed. It cannot be considered as asecondary non-specific consequence of general toxicity.
Reproductive dysfunction in female fish
Impairment of female fish fecundity is also a clear adverse effect observed after TPhP exposure
with a decreased number of eggs and spawning events.
In fact, an inhibition of the ovarian development in female fish (He et al. 2021) and alterationsof ovaries (Li et al., 2019b; He et al., 2021) were observed. Ovary maturation retardation
affected the fecundity of female fish.
TPhP exposure subsequently leads to:- a decrease in egg production (Liu et al., 2013b; Li et al., 2019b, He et al., 2021; Kawashima
et al., 2021),- a decrease in spawning events (Liu et al., 2013b; Li et al., 2018; He et al., 2021).
TPhP induced an increase in circulating E2 concentrations in female zebrafish and female medaka(Liu et al., 2012; Liu et al., 2013b; Liu et al., 2016; Li et al., 2019b; Unpublished study report,
2022). Nevertheless, no change was observed in one study (He et al., 2021). In another study(Li et al., 2019b) carried out on Japanese medaka, a decrease of E2 level was noted after 100day exposure of female larvae (age of fish at exposure initiation), while an increase of E2 wasobserved after 21-day exposure of adult females (age of fish at exposure initiation). The authors
hypothesised that anti-ER activity would retard ovary development, which would further
decrease the E2 level in the long term. To support this hypothesis, the authors showed that the
metabolite 4-OH-TPhP (but not TPhP) was able to inhibit E2 activity in the transgenic medaka
model.
A perturbation in maturation-inducing steroids could cause impairment in oocyte maturation andquality thereby resulting in a decrease in cumulative fecundity.
VTG, which induces oocyte maturation in fish is synthesised in the liver in response to estradiol
stimulation. TPhP induced a down regulation of the expression of VTG1 and VTG2 gene in liver(Liu et al., 2012; Li et al., 2019b) and perturbations of VTG concentrations (Liu et al., 2013b;

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ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHCHe et al., 2021; Kawashima et al., 2021; Unpublished study report, 2022), associated with aconcurrent perturbation of E2. Thus, TPhP activates the estrogenic pathway and/or perturbs thesteroidogenic pathway, which can explain the synthesis perturbation of E2 and VTG after
exposure to TPhP.
The following table reinforces the hypothesis that TPhP disturbs steroid synthesis leading to
impairment in oocyte maturation and subsequently in egg production and spawning byemphasising the consistency of the effects observed in each study. It has to be noted that all
the measurements were performed at the adult stage and the reported concentrations are thosefor which the measured parameters are significant.
Table 16: Dose-response and temporal concordance between the key events and AO for
female fish
[Zebrafish – Japanese Medaka - female] potential dose–response and temporal concordance between
the key events and AO
Dose(µg/L)

Estradiollevel

0.131#

VTG level

Gonad histopathology
impairment↓ (100days)**↓ number of mature oocyte(100 days)

0.299#↓ (100days)***

0.363#↓ number of mature oocyte(100 days)

1.43#

Observed
Adverse effect

Reference
Li et al., 2019b↓ hatching rate

Li et al., 2018

↓ egg production

Li et al., 2019b↓ fertilisation rate

Li et al., 2018

↓ egg production

Li et al., 2019b

1.77#

↑ (21
days)
↓ (100days)

3.01##↑ (73 days)

Unpublished study
report, 2022

↑ (120days)

Liu et al., 2016

5
7.19
7.76##↓ number of mature oocyte(100 days)

Kawashima et al.,2021
Unpublished study
report, 2022
Kawashima et al.,2021
Unpublished study
report, 2022

↓ (21 days)↑ (73 days)

17↓ (21 days)

33.3##↑ (73 days)

76.8##↑ (73 days)↑ (73 days)↓ (21 days)

200↑ (21 days)

1000↑ (120days)↑ (14 days)

Kawashima et al.,2021
Unpublished study
report, 2022

↑ (73 days)#

500↓ egg production↓ fertilised eggs*↓ (21 days)

44.9

80

↓ (21
days)***↓ number of mature oocyte(21 days)↓ hatching rate
↓ fertilisation rate
↓ cumulative eggs
↓ spawning event

He et al., 2021

↓ hatching rate
↓ eggs production↓ spawning event

Liu et al., 2013b

Liu et al., 2016
↓ (14days)**

Liu et al., 201276 (95)

ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC

1000

↑ (21 days)↑ (21 days)↓ hatching rate
↓ egg production↓ spawning event

Liu et al., 2013b* However no significant effect on fertility rate (ratio).
** Transcriptional gene VTG1.
*** Transcriptional gene VTG2.
#
No information on mortality##
Mortality > 10% observed, with mortality > 10% also in the control group
MTC not exceeded for the other studies
Blank cells: parameter not investigated at this concentration

Dose–response and temporal concordance between the key events and AO are often observedat concentration where systemic toxicity was not observed. It cannot be considered as asecondary non-specific consequence of general toxicity.

Conclusion
TPhP shows endocrine activity in fish with adverse effects on fecundity, fertility andsubsequently to reproduction. These adverse effects can be related to the disturbancesin steroid hormone concentrations and impairment of gametogenesis. The resulting
perturbation in maturation-inducing steroids could cause impairment in oocyte andtestis maturation. Changes in fecundity and fertility are apical outcomes, which inform
about potential adverse effects at the population level. Moreover, it is important tonote that several adverse effects on altered reproductive capacity of fish are observedat environmentally relevant levels without concurrent systemic effect.
Overall, based on all available scientific evidence, it can be concluded that TPhP fulfils
the WHO/IPCS definition of an endocrine disruptor:
It shows population relevant adverse effects on fertility and reproduction infish.
It has EAS activity as clearly shown both in vitro and in vivo.
Consistency in the effect and EAS activity observed provides evidence that EAS
modalities
areplausibly
biologicallylinkedtotheadverseeffects.

6. Conclusions on the SVHC Properties
CMR assessment
Not relevant for the identification of the substance as SVHC in accordance with Article 57 (a) to(f) of the REACH Regulation.

PBT and vPvB assessment
Not relevant for the identification of the substance as SVHC in accordance with Article 57 (a) to(f) of the REACH Regulation.

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Assessment under Article 57(f)Summary of the data on the intrinsic/hazardous properties (providing
scientific evidence of probable serious effects to HH and/or ENV)
The available in vitro information demonstrates the capacity of TPhP to competitively bind andactivate the nuclear estrogen receptors ERα and ERβ of several vertebrate species including rat,mouse, fish, chicken, frog and turtle as evidenced by ER transactivation in reporter cell lines (Liuet al., 2012; Kojima et al., 2013; Zhang et al., 2014; Kojima et al., 2016; Li et al., 2018; Ji etal., 2020; Medvedev et al., 2020; Kawashima et al., 2021; Ji et al., 2022). In addition, TPhP can
induce ER-regulated gene expression, and related physiological cell responses (e.g., increased
cell proliferation in Zhang et al. (2014); Krivoshiev et al. (2016); Ji et al. (2020)). Two recent
studies show that TPhP can also bind and activate GPER (Guan et al., 2022 and Ji et al., 2022).
The available H295R assays (Liu et al., 2012; Ji et al., 2022) on human adrenal carcinoma cells
show that TPhP affects steroidogenesis by increasing estrogen levels (17ß-estradiol) and byincreasing expression of genes involved in this pathway like CYP19 and 3β-HSD2. In vivo fishstudies indicate that CYP19A is significantly up-regulated by exposure to TPhP (Liu et al., 2016).
Significant alteration of plasmatic concentrations of E2 and E2/T ratio (more specific observationin females) and E2/11-KT ratio (more specific observation in males) can result from thismodification in the steroidogenesis pathway. The degree of perturbation of circulating steroid
concentrations depends on the fish developmental stage, species and tested concentrations. The
observations of VTG concentrations, that are consistent with perturbation of E2 concentrations,suggest an EAS activity of TPhP in female and male zebrafish, with altered plasmaticconcentration of VTG (Liu et al., 2013b; Unpublished study report, 2022). Therefore, TPhP exerts
an effect on the endocrine balance in fish. It has EAS activity as clearly shown both in vitro andin vivo.
In vivo studies on fish reproduction show an alteration of gametogenesis in both sexes with adisruption of testis (He et al., 2021; Unpublished study report, 2022) and ovary maturation (Liet al., 2019b; He et al., 2021) followed by a decrease in reproductive success in terms of
fecundity: decrease in egg production (Li et al., 2019b; Kawashima et al., 2021), spawning
events (He et al., 2021; Liu et al., 2013b), fertility (impaired hatchability in Liu et al. (2013b);
Li et al. (2018); He et al., (2021)), and fertilisation rate (Li et al., 2018; He et al., 2021).
Therefore, TPhP shows adverse effects on fertility and reproduction in fish, observed in several
studies at levels without concurrent systemic effects.
The consistency between the observed adverse effects and EAS activity provides evidence that
EAS modalities are plausibly biologically linked to the adverse effects. Depending on thedevelopmental stage, species and concentration, antagonist and agonist effects are observed inorganisms, leading in vivo to perturbations of circulating steroid concentrations in most of theanalysed studies.
The effect observed on reproduction in fish (fecundity and fertility) can affect population stability
and is considered as an adverse effect relevant at population level.
Based on all available scientific evidence, it can be concluded that TPhP fulfils the WHO/IPCS
definition of an endocrine disruptor.

Equivalent level of concern assessment
Environment
Triphenyl phosphate shall be identified as a substance of very high concern in accordance with
Article 57(f) of Regulation (EC) 1907/2006 (REACH) because of its endocrine disrupting
properties for which there is scientific evidence of probable serious effects to environment, which

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ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC
gives rise to an equivalent level of concern to those substances listed in points (a) to (e) ofArticle 57 REACH.
In order to assess equivalent level of concern (ELoC), some of the factors identified in ECHA
(2012) to evaluate ELoC for sensitisers, which are considered relevant for the present case, arealso included in the list below:- Characteristics of the effects:• Probable adverse environmental effects related to:o Severity, irreversibility and delayed effects.
•
Broad environmental relevance.
•

Other factors:
o Wide occurrence and environmental distribution.
o Concern related to co-exposure and combined effects.
o Societal concern.
o Is derivation of ‘safe concentration’ possible?

The following description includes the elements relevant to the analysis in an environmental
context.
Probable serious ecotoxicological effects
Severity, irreversibility and delayed effects
TPhP induces adverse effects on reproductive organs and fecundity, and affects the reproductive
ability of fish. Changes in fecundity and fertility are apical outcomes, which inform about
potential adverse effects at the population level, which can lead to serious effects on ecosystems.
Moreover, it is important to note that several adverse effects on altered reproductive capacityof fish are observed at environmentally relevant levels without concurrent systemic effect.
Exposure during the juvenile period of fish may cause gonad histopathological changes, leading
to delayed effects on survival and growth of offspring. There is evidence that a short time
exposure may be sufficient to provoke long-term effects.

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ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC
Broad environmental relevance
Effects on sexual development and reproductive ability via endocrine EAS MoA has a broad
environmental relevance. As data on only a small proportion of the existing species are available,
mainly in fish, potential effects on other organisms remain unknown. Adverse effects are thus
not expected to be restricted to certain taxonomic groups or species. As the main endocrine
systems are known to be largely conserved among vertebrate species in various environments,
it is likely that a wide range of wildlife species with different functions in ecosystems could be
affected.
Wide occurrence and environmental distribution
Several literature studies demonstrate that TPhP can be found in the environment and TPhP has
a widespread use, which leads to a ubiquitous occurrence. TPhP may enter the environment via
emissions from various sources, which is supported by occurrence and monitoring studies(water, sediment, sewage sludge, indoor dust/air). Therefore, environmental species including
predators and vulnerable species (Giulivo et al., 2017; Garcia-Garin et al., 2020; Sala et al.,2021; Sala et al., 2022) are exposed to TPhP. As long as emissions of TPhP do not cease,
exposure of species from the environment cannot be avoided. In addition, human biomonitoringdata demonstrate also the presence of TPhP or its metabolites in human milk (Sundkvist et al.,2010; Kim et al., 2014; Zheng et al., 2021), placenta (Ding et al., 2016; Zhao et al., 2017) andurine (Carignan et al., 2016; Li et al., 2019a) confirming human exposure to this substance fromthe very early stages of life. Moreover, TPhP has also frequently been detected in indoor house
dust samples in Europe, including Belgium and Sweden, and outside Europe, including Japan,
Philippines and USA (Kanazawa et al., 2010; Kim et al., 2013; Marklund et al., 2003; Stapleton
et al., 2009; Van den Eede et al., 2011). Thus, it can be concluded that TPhP reaches diverse
environmental compartments and biota of remote areas including humans.
Concern related to co-exposure and combined effects
TPhPis a constituent or an impurity of several registered substances 15. Thus, mixture effects,
where substances act additively or with synergistic effects, cannot be excluded and this might
impact the threshold of toxicity. Besides, environmental occurrence and human biomonitoringdata (see section 3.2) show that TPhPis detected in the environment, in environmental species
as well as in human fluids together with other organophosphate flame retardants. Typicalexamples are sewage plant effluents and human fluids where TPhPoccurs jointly withorganophosphate flame retardants (Carignan et al., 2016; Ding et al., 2016; Kim et al., 2014;
Li et al., 2019; Sundkvist et al., 2010; Ya et al., 2019; Zhao et al., 2016; Zhao et al., 2017;
Zheng et al., 2021).

Societal concern
In relation to the environment, the impairment of fertility can be an issue regarding species
survival. There is an increasing concern related to the preservation of biodiversity and increasing
evidence that it is threatened due to various causes including global warming and excessive
pressure due to human activities (Jenssen, 2006). EDCs may also contribute to the pressure on
the survival of endangered species (Tubbs and McDonough, 2018). Preserving and restoring
ecosystems and biodiversity is one of the key aims of the European Green Deal (European
Commission, 2019) that is an integral part of the European Commission’s strategy to implement
the United Nation’s 2030 Agenda and the sustainable development goals 16.

1516

https://echa.europa.eu/documents/10162/c7a3c8b0-3fd3-3a4a-e791-2b4ae6a696d5
https://sustainabledevelopment.un.org/post2015/transformingourworld

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ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHCIs derivation of ‘safe concentration’ possible?
Endocrine regulation, which is set up during critical life stages in vertebrates, is a complex
feedback process. Any disturbance of this regulation during transient but vulnerable life stages
can lead to irreversible effects for the entire lifetime or even in the following generations which
will also be dependent on the organism group. Moreover, based on the available ED specific test
guidelines, it is difficult to assess the latency of the effects. Therefore, prediction of future effects
and derivation of safe concentration for the environment is associated with large uncertainties.
Thus, effects may be overlooked, not expressed or equivocal. For TPhP, for example, no data isavailable for most of the trophic levels, which makes it difficult to derive a safe exposure levelin the environment. Another reason may be that low effect concentrations are difficult todetermine definitively, as effects may only be observed in certain life stages or time windows.
Additionally, seasonal effects may lead to difficulty in predicting the impact on the development
of different groups of organisms.

The ELoC elements are summarised in Table 16 below.

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Table 18: ELoC summary reporting
Adverse effects on reproduction
Probable serious
effects?*

Overall conclusion▪ Adverse effects on reproductive organs of fish exposed to TPhP.

YES▪ Adverse effect on fecundity and reproductive ability in fish exposed
to TPhP.

▪ Pattern of ED-related effects associated withserious dysfunction in fish (fecundity, reproductive
ability, gonad histopathological effects).

▪ Several adverse effects on altered reproductive capacity of fishobserved at environmentally relevant levels without concurrent
systemic effect.
▪ Delayed andirreversibility ofeffects?

▪ Exposure during juvenile period of fish may cause gonad
histopathological changes, leading to delayed effects on survival andgrowth of offspring.
▪ There is evidence that a short time TPhP exposure may be sufficient
to provoke long-term effects on survival and growth of offspring.
▪ Irreversible effects during the entire lifetime or even in the following
generations are expected following disruption of endocrine
regulation during transient but vulnerable life stages.

▪ Broad environmental
relevance▪ Broad environmental relevance due to the effects observed on
sexual development and reproductive ability via endocrine EAS MoA.

YES
▪ Exposure during the juvenile period of fish has
been shown to have consequences in relation to
ED-related effects later in life.
▪ Short time TPhP exposure may be sufficient toprovoke long-term effects on survival and growthof offspring.
▪ Due to conservation of the reproductive endocrine
system in a wide range of taxa in different
ecosystems, other species or taxa can be impacted
as well.
YES▪ ED MoA with broad environmental relevance.

▪ As data on only a small proportion of the existing species areavailable, mainly fish, potential effects on other organisms remain
unknown.
▪ It is very likely that a wide range of wildlife species with different
functions in ecosystems could be affected.

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ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC
Wide occurrence andenvironmental
distribution▪ Widespread use of TPhP, which leads to an ubiquitous occurrence.

YES▪ Environmental species including predators and vulnerable species(fin whale) can be exposed more or less continuously to TPhP and
exposure can thus not be avoided.

▪ Wide occurrence and environmental distribution.

▪ Concern supported by occurrence and environmental monitoringdata (water, sediment, sewage sludge, indoor dust/air) and humanstudies demonstrating the presence of TPhP or its metabolites inhuman milk, placenta and urine.
Potential to cause
combined effects (coexposure)▪ TPhP is a constituent or an impurity of several registeredsubstances17.
▪ Mixture effects cannot be excluded and may impact the threshold of
toxicity.

YES▪ Combined exposure cannot be avoided and thus
combined effects cannot be ruled out.

TPhP and organophosphate flame retardant co-exposure have been
documented in sewage plant effluents and in human fluids.
Societal concern?

▪ Fertility impairment can be an issue regarding environmental
species survival.

YES▪ Major societal environmental concerns.

▪ Increasing concern about biodiversity preservation.
▪ EDC may also contribute to the challenge of survival of endangered
species.
Uncertainties inderiving safe
concentration limits▪ Endocrine regulation, which is set up during critical life stages invertebrates, is a very complex feedback process.
▪ This complex ED feedback process prevents to predict potential
future effects and thus safe exposure levels for the environment.

YES▪ Derivation of safe concentration associated withlarge uncertainties.

▪ For TPhP, data is not available for all trophic levels, which makes it
difficult to derive a safe exposure level in the environment.

*This factor is intended to discuss the severity of the effects and not their probability

17

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Summary of the ELoC assessment
Triphenylphosphate (TPhP) fulfils the WHO/IPCS definition of an endocrine disruptor relevant
for the environment. The very high concern raised by this property is substantiated by theseverity and irreversibility of the effects observed on organisms. These effects are relevant at
the population level and may have long term consequences. A large variety of species may be
adversely affected and there are difficulties to quantify a safe level of exposure with regard to
the endocrine mediated effects.
In conclusion, there is scientific evidence that TPhP causes probable serious effects to theenvironment due to its endocrine disrupting properties, which give rise to an equivalent levelof concern to those of other substances listed in points (a) to (e) of Article 57 of the REACH
Regulation.
TPhP is proposed to be identified as a substance of very high concern in accordance with Article
57(f) of Regulation (EC) 1907/2006 (REACH) because it is a substance with endocrine
disrupting properties for which there is scientific evidence of probable serious effects to theenvironment, which give rise to an equivalent level of concern to those of other substances
listed in points (a) to (e) of Article 57 REACH.

Conclusion on the Article 57(f) assessment
TPhP is proposed to be identified as a substance of very high concern in accordance with Article
57(f) of Regulation (EC) 1907/2006 (REACH) because it is a substance with endocrine
disrupting properties for which there is scientific evidence of probable serious effects to theenvironment, which give rise to an equivalent level of concern to those of other substances
listed in points (a) to (e) of Article 57 REACH.
The available in vitro information demonstrates the capacity of TPhP to competitively bind andactivate nuclear estrogen receptors ERα and ERβ of several vertebrate species including rat,mouse, fish, chicken, frog and turtle as evidenced by ER transactivation in reporter cell lines(Liu et al., 2012; Kojima et al., 2013; Zhang et al., 2014; Kojima et al., 2016; Li et al., 2018;
Ji et al., 2020; Medvedev et al., 2020; Kawashima et al., 2021; Ji et al., 2022). In addition,
TPhP can induce ER-regulated gene expression, and related physiological cell responses (e.g.,increased cell proliferation in Zhang et al., 2014; Krivoshiev et al., 2016; Ji et al., 2020). Tworecent studies show that TPhP can also bind and activate GPER (Guan et al., 2022; Ji et al.,2022). The available H295R assays (Liu et al., 2012; Ji et al., 2022) on human adrenal
carcinoma cells show that TPhP affects steroidogenesis by increasing estrogen levels (17ßestradiol) and by increasing expression of genes involved in this pathway like CYP19 and 3βHSD2. In vivo fish studies indicate that CYP19A is significantly up-regulated by exposure to
TPhP (Liu et al., 2016). Significant alteration of plasmatic concentrations of E2 and E2/T ratio(more specific observation in females) and E2/11-KT ratio (more specific observation in males)
can result from this modification in the steroidogenesis pathway. The degree of perturbation of
circulating steroid concentrations depends on the fish developmental stage, species and tested
concentrations. The observations of VTG concentrations, that are consistent with perturbationof E2 concentrations, suggest an EAS activity of TPhP in female and male Zebrafish, with alteredplasmatic concentration of VTG (Liu et al., 2013b; Unpublished study report 2022). Therefore,
TPhP exerts an effect on the endocrine balance in fish. It has EAS activity as clearly shown bothin vitro and in vivo.
In vivo studies on fish reproduction show an alteration of gametogenesis in both sex with adisruption of testis (He et al., 2021; Unpublished study report 2022) and ovary maturation (Liet al., 2019b; He et al., 2021) followed by a decrease in reproductive success in terms of
fecundity : decrease in egg production (Li et al., 2019b; Kawashima et al., 2021), spawning
events (He et al., 2021;Liu et al., 2013b), fertility (impaired hatchability in Liu et al., 2013b;

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ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC
Li et al., 2018;He et al., 2021), and fertilisation rate (Li et al., 2018; He et al., 2021). Therefore,
TPhP shows adverse effects on fertility and reproduction in fish, observed in several studies atlevels without concurrent systemic effect.
The consistency between the observed adverse effects and EAS activity provides evidence that
EAS modalities are plausibly biologically linked to the adverse effects. Depending on thedevelopmental stage, species and concentration, antagonist and agonist effects are observedin organisms, leading in vivo to perturbations of circulating steroid concentrations in most of
the analysed studies.
The effect observed on reproduction in fish (fecundity and fertility) can affect population
stability and is considered as an adverse effect relevant at population level.
Based on all available scientific evidence, it can be concluded that TPhP fulfils the WHO/IPCS
definition of an endocrine disruptor. The very high concern raised by this property is
substantiated by the severity and irreversibility of the effects on organisms and populations
that may have long term consequences, the large variety of species that may be adversely
affected and the difficulties to quantify a safe level of exposure with regard to the endocrine
mediated effects.
In conclusion, there is scientific evidence that TPhP causes probable serious effects to theenvironment due to its endocrine disrupting properties, which give rise to an equivalent levelof concern to those of other substances listed in points (a) to (e) of Article 57 of the REACH
Regulation.

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ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHCPart II
7. Registration and C&L notification status
Registration status
Table 19 Registration status

From the ECHA dissemination site18☒ Full registration(s)Registrations(Art. 10)
Intermediate registration(s)(Art. 17 and/or 18)

CLP notification status
Table 20: CLP notifications
CLP Notifications19
Number of aggregated notifications

25

Total number of notifiers

1440

Substance specific entry on the dissemination site: Registration Dossier - ECHA (europa.eu) (accessed 14-112023).
19
C&L Inventory database, http://echa.europa.eu/web/guest/information-on-chemicals/cl-inventory-database(accessed 14-11-2023).
18

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ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC

8. Total tonnage of the substance
Table 21: Tonnage status

Total tonnage band for the registeredsubstance (excluding the volume
registered under Art 17 or Art 18) 20
Tonnage information from public sources
other than registration dossiers (ifavailable)21≥ 100 to < 1 000 t/pa

No information

9. Information on uses of the substance
TPhP is used as a flame retardant and plasticiser in polymer formulations, adhesives andsealants, cosmetics and personal care products. TPhP is used by consumers, in articles, byprofessional workers (widespread uses), in formulation or re-packing, at industrial sites and in
manufacturing. TPhP is present as an impurity in many other organophosphate flame
retardants or as a constituent of this family of compounds.
Other releases to the environment of this substance are likely to occur from: indoor use andoutdoor use resulting in inclusion into or onto materials (e.g. binding agent in paints andcoatings or adhesives).
The following information is extracted from the ECHA dissemination website.
Table 22: Uses
Use(s)

Uses asintermediate

Formulation
or repacking-

Formulation of plastic (compounding)and rubber preparations (compounding),
including coatings and adhesive
products.

Registereduse

Use likely to be in
the scope ofAuthorisation

Yes

No

Yes

Yes

Yes

Yes

Formulation of flame retardant/plasticiser
preparations and cosmetics.
Uses atindustrial
sites

Production of plastic and rubber articles(conversion)

ECHA’s dissemination site https://echa.europa.eu/fr/information-on-chemicals/registered-substances//disreg/substance/100.003.739 (accessed 14 11 2023, last updated 21-07-2022).
20

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ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC
Uses byprofessional
workers

Use of adhesives and sealants, outdoor.

Yes

Yes

Yes

Yes

Yes

No

Use of adhesives and sealants, indoor.
Use as laboratory chemicals.
Use of adhesives and sealants, outdoor.
Use of adhesives and sealants, indoor.

Consumeruses

Use of cosmetic products containing
triphenyl phosphate. Other release to theenvironment of this substance is likely tooccur from: indoor use and outdoor use
resulting in inclusion into or onto
materials (e.g. binding agent in paints
and coatings or adhesives).
Release to the environment
substance can occur from:

Articleservice life

of

this

Waste
stage:
hazardous
waste
incineration, municipal and industrial.
- Waste stage: shredding.
Waste
stage:
incineration.

municipal

waste- Municipal landfill.

10. Information on structure of the supply chain
No information available.

11. Additional information
No additional information available.

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ANNEX XV – IDENTIFICATION OF TRIPHENYL PHOSPHATE AS SVHC

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