Tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate

Endocrine disrupter testing in aquatic vertebrates – in vivo

Administrative data

Endpoint:

fish: other

Remarks:

Fish sexual development test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6th April 2021 - 16th February 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD TG 234 (Fish Sexual Development Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate

EC Number:

222-020-0

EC Name:

Tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate

Cas Number:

3319-31-1

Molecular formula:

C33H54O6

IUPAC Name:

tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate

Details on test material:

- Name of test material (as cited in study report): DIPLAST TM
- Physical state: colourless liquid
- Lot/batch No.: TM0937
- Expiration date of the lot/batch: 1 year from receipt
- Storage condition of test material: room temperature in a sealed bag with silica gel
- Purity: 98.43%

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.2, 0.5, 1.0, 2.3, 5.0 µg/L measured on days 0, 8, 11, 14, 21, 27, 35, 42, 49, 56 and 64 of exposure.
- Sampling method: Water samples collected from the mid-point of the test vessels into 250 mL glass bottles.
- Sample storage conditions before analysis: Samples were analysed on the day of collection.

Test solutions

Vehicle:

yes

Remarks:

Solvent - acetone

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock concentrate of 60 mg/L was prepared weekly by dissolving the test item in acetone. This was used to prepare the acetone stock solutions (nominal 2.4, 6.0, 12.0, 27.6 and 60.0 mg/L) for dosing. A continuous flow-through dilutor system was used to dilute the stock solutions by 12000 to generate the required exposure concentrations (nominal 0.2, 0.5, 1.0, 2.3, 5.0 µg/L).
- Eluate: Not applicable
- Differential loading: Not applicable
- Controls: The controls were a dechlorinated dilution water control, without addition of acetone or test item, and a solvent control which received the same conentration of acetone as the test solutions (nominal 80 µL/L).
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Acetone
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 80 µL/L
- Test concentration separation factor: 2.2
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): None
- Other relevant information: None

Test organisms

Aquatic vertebrate type:

fish

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Strain:London wild-type
- Source:Brood stock: University of Sheffield
- Life stage: Embryos
- Age at study initiation (mean and range, SD): <12 hours fertilisation (all embryos)
- Length at study initiation (length definition, mean, range and SD): not determined
- Weight at study initiation (mean and range, SD): not determined
- Method of breeding: Spawning groups (n=16) of 3males + 3females alllowed to spawn naturally
- Pre-exposure reproductive information: Brood stock was maintained for >3months in the test laboratory. Fed at least twice daily with frozen brine shrimp and Artemia. No treatment for disease. No mortality in the week prior to the test.

ACCLIMATION
- Acclimation period: >3 months
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food during acclimation: Frozen brine shrimp and Artemia, fed to satiation.
- Feeding frequency during acclimation: At least twice daily
- Health during acclimation (any mortality observed): No mortalities

QUARANTINE (wild caught)
- Duration: not applicable
- Health/mortality: not applicable

FEEDING DURING TEST
- Food type: Dry fry food (ZM-000 and ZM-100), frozen rotifers, Artemia sp.
- Amount: Fed to satiation
- Frequency: 2-3 times daily to 32 days post hatch, 2-time daily from 33 days post hatch.

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Numbers of parental fish (i.e. of females used to provide required number of eggs): 96 fish divided over 16 spawning tanks (3 males + 3 females per tank)
- Method of collection of fertilised eggs: Spawning trays placed in spawning tanks to collect the eggs.
- Subsequent handling of eggs: Eggs pooled, then transferred, in batches of 5, using a wide-bore pipette to egg incubation cups (15 embryos/cup) which were then suspended in the test vessels. Two incubation cups per vessel.
- Subsequent handling of juvenile fish: Fry released from incubation cups once 90% of controls had hatched.

POST-HATCH FEEDING
- Start date: From hatch
- Type/source of feed: Dry fry food (ZM-000 and ZM-100), frozen rotifers, Artemia sp.
- Amount given: Fed to satiation
- Frequency of feeding: 2-3 times daily to 32 days post hatch, 2-time daily from 33 days post hatch.

Study design

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

64 d

Post exposure observation period:

Not applicable

Test conditions

Hardness:

282 - 300 mg/L of CaCO3; measured in one replicate test vessel per treatment at the start and end of the test

Test temperature:

26.2 - 27.4 °C, temperature was continuously monitored in one control vessel using a data logger. Temperatures in individual test vessels were determined twice weekly during the test.

pH:

7.94 - 8.34; pH was measured twice weekly in each test vessel throughout the test

Dissolved oxygen:

87.4 – 99.7 %Saturation; DO was measured twice weekly in each test vessel throughout the test

Salinity:

N/A freshwater

Conductivity:

618-625 µS/cm; measured twice weekly in each test vessel throughout the test

Nominal and measured concentrations:

Nominal concentrations were 0.2, 0.5, 1.0, 2.3, 5.0 µg/L. Nominal concentrations were achieved prior to introduction of the test organisms (between 83 and 111%) but became increasingly variable (>20% variability) over time due to the hydrophobic nature of the test item and it's tendancy to partition to organic matter. Mean measured concentrations (0.35, 0.65, 1.37, 2.98, 9.38 µg/L) over the full duration of the test were therefore used in analysis of the results.

Details on test conditions:

TEST SYSTEM
- Test vessel: glass vessel
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: Glass, 300x200x250mm, with 50 mm headspace, and 10.5L fill volume
- Aeration: None
- Type of flow-through (e.g. peristaltic or proportional diluter): Peristaltic
- Renewal rate of test solution (frequency/flow rate): 20.6 tank volume changes per day
- No. of organisms per vessel: 30
- No. of organisms per mL or well: 2.9 fish per litre
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Vehicle control performed: yes
- No. of vessels per vehicle control (replicates): 4
- Biomass loading rate: not determined
- Stocking density: <0.23 g/L
- No. of independent and valid run (solutions and spawn): 1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dechlorinated mains water
- Total organic carbon: 0.43 mg/L
- Particulate matter: <2.0 mg/L Total suspended solids
- Metals: Aluminium (<20 µg/L), Arsenic (0.1 µg/L), Cadmium (<0.08 µg/L), Chromium (<0.3 µg/L), Cobalt (<0.08 µg/L), Copper (12 µg/L), Iron (<3 µg/L), Lead (< 0.04 µg/L), Mercury <0.04 µg/L), Nickel (<0.3 µg/L), Silver (< 0.01 µg/L), Zinc (2 µg/L)
- Pesticides: <0.03 µg/L
- Chlorine:<0.05 mg/L
- Alkalinity: not determined
- Ca/mg ratio: not determined
- Culture medium different from test medium: no
- Intervals of water quality measurement: 3 times per year

RANGE FINDING STUDY
- Test concentrations: 0.05, 0.5 and 5 µg/L
- Results used to determine the conditions for the definitive study: No evidence for a negative effect of TOTM on zebrafish (exposed from <12 h post fertilisation through to 20 days post hatch) survival (pre-and post-hatch), mean body weights and standard lengths.

Reference substance (positive control):

yes

Remarks:

solvent - acetone

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

3 d

Dose descriptor:

NOEC

Effect conc.:

> 9.38 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

time to hatch

Remarks on result:

other: Time to first hatch; data compared to solvent control

Effect concentrations 2

Duration:

4 d

Dose descriptor:

NOEC

Effect conc.:

> 9.38 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

number hatched

Remarks on result:

other: % survival to hatch; data compared to solvent control

Effect concentrations 3

Duration:

5 d

Dose descriptor:

NOEC

Effect conc.:

> 9.38 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

time to hatch

Remarks on result:

other: Time to first hatch; data compared to solvent control

Effect concentrations 4

Duration:

60 d

Dose descriptor:

NOEC

Effect conc.:

> 9.38 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: % survival post-hatch; data compared to solvent control; statistical effect detected at 0.35, 0.65 and 1.37 µg/L but no effect at concentrations of 2.98 and 9.38 µg/L

Effect concentrations 5

Duration:

64 d

Dose descriptor:

NOEC

Effect conc.:

> 9.38 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: %cumulative survival; data compared to solvent control; statistical effect detected at 0.35, 0.65 and 1.37 µg/L but no effect at concentrations of 2.98 and 9.38 µg/L

Effect concentrations 6

Duration:

64 d

Dose descriptor:

NOEC

Effect conc.:

> 9.38 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

length

Remarks on result:

other: Standard body length; data compared to solvent control; statistical effect detected at 1.37 µg/L but no effect at higher concentrations (2.98 and 9.38 µg/L)

Effect concentrations 7

Duration:

64 d

Dose descriptor:

NOEC

Effect conc.:

> 9.38 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

wet body weight

Remarks on result:

other: data compared to solvent control; statistical effect detected at 1.37 µg/L but no effect at higher concentrations (2.98 and 9.38 µg/L)

Effect concentrations 8

Duration:

64 d

Dose descriptor:

NOEC

Effect conc.:

> 9.38 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

genetic/phenotypic sex ratio

Remarks on result:

other: Proportion of phenotypic males; data compared to solvent control

Effect concentrations 9

Duration:

64 d

Dose descriptor:

NOEC

Effect conc.:

> 9.38 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

genetic/phenotypic sex ratio

Remarks on result:

other: Proportion of phenotypic females; data compared to solvent control

Effect concentrations 10

Duration:

64 d

Dose descriptor:

NOEC

Effect conc.:

> 9.38 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

genetic/phenotypic sex ratio

Remarks on result:

other: Proportion of undifferentiated fish; data compared to solvent control

Effect concentrations 11

Duration:

64 d

Dose descriptor:

NOEC

Effect conc.:

> 9.38 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

genetic/phenotypic sex ratio

Remarks on result:

other: Proportion of intersex fish; data compared to solvent control

Effect concentrations 12

Duration:

64 d

Dose descriptor:

NOEC

Effect conc.:

> 9.38 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

vitellogenin level

Remarks on result:

other: Vitellogenin level in males; data compared to solvent control

Effect concentrations 13

Duration:

64 d

Dose descriptor:

NOEC

Effect conc.:

> 9.38 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

vitellogenin level

Remarks on result:

other: Vitellogenin level in females; data compared to solvent control

Details on results:

See table "Any other information on results incl. tables"

Any other information on results incl. tables

Summary of mean values (across 4 replicates) for each concentration level. The value in brackets represents the CoV for the survival, hatch and body size data. For the vitellogen data and %males, SD values are provided in brackets. **denotes a significant difference detected relative to the solvent control (p <0.05)

Concn (µg/L)	%survival to hatch	Time to first hatch	Time to last hatch	%survival post-hatch	%cumulative survival	Standard body length	Wet body weight	%males	%females	%undifferentiated	%intersex	Male VTG	Female VTG
0 (dilution ctrl)	99 (1.68)	3 (0.00)	4.5 (12.83)	85 (2.12)	84 (1.98)	17 (0.83)	75.1 (2.67)	49.5	48.5	2.0	0.0	379 (206)	162477 (64260)
0 (solvent ctrl)	96 (3.33)	3 (0.00)	4.3 (11.76)	91 (3.28)	87 (0.00)	17.8 (0.79)	80.3 (4.10)	58.7	41.3	0.0	0.0	412 (165)	185022 (158325)
0.35	98 (1.96)	3 (0.00)	4.5 (12.83)	80 (8.11)**	79 (9.34)**	17.5 (2.89)	78.8 (7.86)	57.4	41.5	1.0	0.0	362 (181)	139309 (112103)
0.65	98 (1.96)	2.5 (23.09)	4 (0.00)	79 (4.03)**	78 (4.12)**	17.9 (1.41)	82 (4.63)	63.4	35.5	1.0	0.0	562 (591)	603027 (998498)
1.37	95 (4.53)	3 (0.00)	4.3 (11.76)	79 (10.26)**	75 (5.74)**	17.1 (0.74)**	71.3 (3.06)**	61.1	35.6	1.0	1.0	254 (44)	41416 (33624)
2.98	95 (3.51)	3 (0.00)	4 (0.00)	87 (3.36)	83 (2.02)	17.4 (1.82)	76.5 (2.26)	52.5	46.5	0.0	1.0	338 (150)	94811 (58227)
9.38	95 (4.53)	2.5 (23.09)	4 (0.00)	92 (6.68)	88 (3.65)	17.9 (1.38)	82.4 (2.33)	67.6	31.4	1.0	0.0	356 (253)	88990 (38677)

The acceptance criteria for this study, based on OECD guideline 234, were for:

Performance criteria	Assessment
Dissolved oxygen to remain above 60% air saturation value	Pass
Water temperature to be maintained within the temperature range 27 ± 2°C	Pass
Water temperature to not differ by more than 1.5°C between test vessels or between successive days	Pass
Measured test concentrations to be maintained within ± 20% of the mean measured values per treatment level	Fail*
For the pooled replicates of the controls; pre-hatch survival > 80%	Pass
For the pooled replicates of the controls; post-hatch survival ≥ 70%	Pass
At the end of the test, for pooled replicates of the controls, fish weights > 75 mg and standard lengths > 14 mm	Pass
The sex ratio (% males), for pooled replicates of the controls to be within the range 30-70%	Pass

*as measured test concentrations varied by more than 20% (29 to 68% variation observed) per treatment level the results are analysed and reported using mean measured concentrations. Failure of this performance criteria is not considered to have impacted the integrity of the study.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

7 of the 8 validity criteria fulfilled. See above for the one that failed.

Conclusions:

Aqueous exposure of early life stages of zebrafish to TOTM at concentrations up to 9.38 µg/L does not adversely affect health. There is no evidence for an effect of TOTM on the endocrine system (as assessed through determinations of phenotypic sex ratio and vitellogenin levels), indicating that TOTM does not possess endocrine disrupting properties.

Executive summary:

The purpose of this study was to assess early life-stage effects and potential adverse consequences of tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate (TOTM) on sexual development. Zebrafish were exposed from embryo (<12 h post fertilisation) through to 60 days post-hatch to 5 concentrations of the test item (mean measured concentrations: 0.35, 0.65, 1.37, 2.98 and 9.38 µg/L) a dilution control and an acetone solvent control under flow-through conditions. Mean hatch success, time to last hatch, post-hatch survival and cumulative survival (pre- and post-hatch) in the (solvent) control was 96%, 4.0 days, 91% and 87%, respectively. There was no effect of exposure to TOTM on survival or time to hatch. Statistical analysis indicated an apparent effect of TOTM (at concentrations of 0.35, 0.65 and 1.37 µg/L) exposure on post-hatch survival (and cumulative survival). However, a negative trend between concentration and survival was not observed, and survival in the higher concentrations (2.98 and 9.38 µg TOTM/L) was comparable to that observed in the controls indicating that TOTM exposure does not impact fish survival. Mean wet weights and total body lengths, at 60 days post hatch, were 80.3 mg and 17.8 mm, respectively, for the solvent control. Statistical analysis indicated an apparent effect of TOTM on fish body size at 60 days post hatch in the intermediate exposure group (1.37 µg TOTM/L). However, there was no evidence for a relationship between concentration and magnitude of effect and fish body sizes were comparable in the higher concentrations (2.98 and 9.38 µg TOTM/L) to body sizes observed in the controls, suggesting that TOTM exposure does not impact fish growth.
At the end of the test, the mean sex ratio in the (solvent) control was determined to be 59% male, 41% female. There was no evidence for an effect of exposure to TOTM on the proportion of male, female, undifferentiated or intersex fish (NOEC >9.38 µg/L). Mean vitellogenin titres (in the head/tail homogenates) were 412 and 185022 ng/mL for the phenotypic male and female fish respectively. There was no evidence for an effect of exposure to TOTM on male or female vitellogenin concentrations (NOEC >9.38 µg/L). Results of this study lead to the conclusion that TOTM does not possess endocrine disrupting properties.