D-Glucitol, 1,5-anhydro-1-C-[3-[[5-(4-fluorophenyl)-2-thienyl]methyl]-4-methylphenyl]-, 2,3,4,6-tetrakis(2,2-dimethylpropanoate), (1S)-

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 JUL 2021 - 16 AUG 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

Version / remarks:

2013

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance Document on Aqueous-phase Aquatic Toxicity Testing of Difficult Test Chemicals, OECD series on testing and assessment number 23, February 08, 2019

Version / remarks:

2019

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- lot/batch number of test material: I20IB2949
- Purity; 98.9%, retest date 19-SEP-2022, no correction factor required
- Expiration date: 19 September 2022 (retest date)
- Purity test date: 2 November 2020 (certificate of analysis release date)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability in water and acetone: not indicated
- Solubility in water: < 0.01 g/L at 21.5° pH: 7.7
- Solubility in acetone: 150 g/L

Analytical monitoring:

yes

Details on sampling:

- Sampling method: Samples for possible analysis were taken from all test concentrations and the controls. Samples were taken one day before the start of exposure to check the functioning of the system, at the start, and at weekly intervals (i.e. on day 7, 14, 21, and 28) of exposure. Samples of the stock solutions were taken on day -2, 7, 14, 21 and 28. The volume of samples was 1.0 mL (day -2, -1, 0, 7, 14) and 2.0 mL (day 21).

- Sample storage conditions before analysis:
Samples were transferred to the analytical laboratory at the Test Facility and analysed on the day of sampling.
Samples of the stock solution of day -2 were stored in a freezer (set to maintain -15°C) until analysis at the analytical laboratory of the Test Facility.
Additionally, reserve samples of 1.0 mL (day -2, -1, 0, 7, 14) and 2.0 mL (day 21) were taken from all stocks and test solutions for possible analysis. If not already used, these samples were stored in a freezer (≤15°C) for a maximum of three months after delivery of the draft report.

Vehicle:

yes

Remarks:

acetone

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stock solutions of 300 µg/mL were prepared in acetone (Merck, Darmstadt, Germany) three times a week. Lower stock solutions, i.e. 3.0, 9.4, 30 and 94 µg/mL were prepared by diluting the highest stock with acetone. All stock solutions were clear and colourless.
- Controls:
Blank control: test medium without test item or other additives
Solvent control: test medium without test item but with the additive used in the treatment of the stock solutions
- Chemical name of vehicle: acetone
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 0.1 ml/L
- Test concentration separation factor: 3.2
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): All stock solutions were clear and colourless

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: Fathead minnow
- Strain: Rafinesque
- Source: In-house culture

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Ratio male/female: 1:2. Males and females are put together in spawning tanks and spawning starts the following day approximately 1 to 2 hours after lights have been switched on.
- Method of collection of fertilised eggs: the spawning tank is equipped with substrate (pvc-tube), which enables collection of the fertilised eggs.

FEEDING
- Feeding brood stock: Frozen brine shrimp Nauplii and pelleted fish food (SDS 400, Coppens International bv, Helmond, The Netherlands)
- Embryonic phase: no feeding.
- Larvae and juvenile fish: Brine shrimp Nauplii 24 or 48 hours old. Food was supplied ad libitum.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

32 d

Remarks on exposure duration:

32 days post-fertilization

Hardness:

214-250 mg/L (as CaCO3).

Test temperature:

24.5-25.4 °C (measured in test vessels)
24.6-25.5 °C (measured continuously in control vessel)

pH:

7.3-8.0

Dissolved oxygen:

5.8-8.9 mg O2/L

Nominal and measured concentrations:

Nominal concentrations: 0.30, 0.94, 3.0, 9.4 and 30 µg/L
Mean measured concentrations: 0.36, 0.98, 3.9, 9.5 and 36 µg/L.

Analysed samples from the stock solution on a weekly basis showed that the measured concentrations were generally in agreement with the nominal concentrations of the stock solutions, ranging between 88 and 110% relative to the respective targets.
Analyses of samples taken from the nominal concentrations on a weekly basis showed that the measured concentrations were not stable during the exposure period. The concentrations ranged from 58 to 246% relative to the respective targets in test medium. A reason for this instability could not be identified. The dosing system operated according to expectation and stock solutions were correctly prepared. Based on this, the results were accepted and average exposure concentrations were calculated and used for the further study evaluation. The mean measured concentrations represented 101-130% of the targets. The coefficient of variation was between 20 and 54%.

Details on test conditions:

TEST SYSTEM
- Test vessel: Stainless steel vessels (~1.7 L)
- Renewal rate of test solution (frequency/flow rate): flow-through with continuous renewal of test media
- Dosing system: Exact volumes of the test item stock solutions in acetone were dosed with syringes via a computer-controlled system consisting of five dispensers (Gilson). The dosed volumes of the stock entered a mixing flask separately from the dilution water supply (test medium). The dilution water was supplied applying flow meters at a constant rate of 5 litres per hour. In the mixing flask the dosed volume and the dilution water were mixed under continuous stirring. Peristaltic pumps, set at a rate of 1 litre per hour, were used to divide the contents of the mixing vessels continuously and equally over four replicate test vessels containing the eggs/fish larvae/juvenile fish. The flow meters were calibrated before the start of the exposure and weekly thereafter.
- No. of fertilized eggs/embryos per vessel: 80 fresh and healthy fertilised fathead minnow eggs per test group
- Introduction eggs: ca. 1.5h after the cleavage of the blastodisc commenced
- No. of vessels per concentration (replicates): 4, each containing 20 eggs
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: RO-water + salts (analytical grade) of CaCl2.H2O, MgSO4.7H2O, NaHCO3, KCl
- Total organic carbon: The concentration of TOC in a sample taken from the test medium was 1.1 mg/L and as such complied with the value recommended by the guideline (i.e. <2 mg TOC/L).
- Intervals of water quality measurement:
Oxygen, pH and temperature: at the start of the test, at weekly intervals and at the end of the test in all replicates. In addition, temperature was recorded continuously in one of the control replicates.
Hardness: At the start and end of the test in all test groups.
TOC: At the start of the test in blank medium.
Light intensity: at the start and end of the test, measured at the edges and the centre part of the experimental set-up and at the approximate height of the centre of the test vessels.

OTHER TEST CONDITIONS
- Photoperiod: 16 h photoperiod daily, between 605 and 710 lux
- Light intensity: did not exceed 1,000-1,500 lux or 15-20 µE/m2/s

VEHICLE CONTROL PERFORMED: yes

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Stage of embryonic development: At the beginning of exposure from all eggs at least a representative sample of eggs.
Hatching and survival: Daily from nominal day 1, numbers of hatched larvae and dead embryos, larvae and juvenile fish were recorded. Dead embryos, larvae and juvenile fish were removed directly after recording. The criteria for death were the following: Eggs: particularly in the early stages, a marked loss of translucency and change in coloration, caused by coagulation and/or precipitating of protein, characterised by a white opaque appearance. Embryos: Absence of body movement and /or absence of heartbeat. Larvae and juvenile fish: immobility and/or absence of respiratory movement and/or absence of heartbeat and/or white opaque coloration of the central nervous system and/or lack of reaction to mechanical stimulus.
Abnormal appearance: Daily, abnormalities were recorded, e.g. hyperventilating, uncoordinated swimming, atypical quiescence and atypical swimming behaviour.
Body weight: At the end of the test, all surviving fish were weighed on a replicate basis (blotted dry weight).
Body length: At the end of the test, individual lengths from the surviving fish were measured.

Reference substance (positive control):

no

Key result

Duration:

32 d

Dose descriptor:

LOEC

Effect conc.:

9.5 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

weight

Key result

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

3.9 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

weight

Details on results:

- Embryonic survival: 6d-LOEC > 36 µg/L, 6d-NOEC = 36 µg/L
- Post-hatch survival: 32d-LOEC > 36 µg/L, 32d-NOEC = 36 µg/L
- Growth (weight): 32d-LOEC = 9.5 µg/L, 32d-NOEC = 3.9 µg/L
- Growth (length): 32d-LOEC > 36 µg/L, 32d-NOEC = 36 µg/L

- Comparison of control treatments: Statistical comparisons showed that there was no significant difference between any of the endpoints in the blank and the solvent control. Subsequently, the pooled control was used to evaluate the potential effects of test item exposure.

Embryonic survival:
- Hatching commenced on day 3 and was completed on day 6 of exposure.
- The overall survival of embryos at the end of hatching was 100% for the blank- and solvent control.
- The overall survival of embryos at the end of the hatching was between 96 and 100% in the test concentrations.
- Hatching success: No statistically significant differences were observed at any of the concentrations tested when compared to the pooled control.

Post-hatch survival:
- The mean post-hatch larval survival was 96% for the blank control and 97% for the solvent control.
- The post-hatch survival in the test groups ranged between 92 and 99% at the end of the test.
- Post-hatch survival: No statistically significant differences on post-hatch survival were observed at any of the concentrations tested when compared to the pooled control.
- Developmental defects were observed sporadically in the control groups and the test concentrations during the exposure period. Most prominent symptoms included malformations of the body and swim bladder malformations. Statistical analysis was not performed on abnormalities but based on the observations made during the test it appears that the severity and incidence of abnormalities was not dose related.

Growth:
- Mean body length of the larvae at the end of the test was 18 to 19 mm in the blank control and the solvent control, respectively
- Mean body weight of the larvae at the end of the test was 55 and 57 mg in the blank control and the solvent control, respectively.
- Growth: The test substance caused no clear dose-related reduction in body weight. Statistical analysis indicated a statistically significant effect at the two highest concentrations but not at the lower concentrations. The reduction of body weight at the concentration of 36 μg/L was lower than the reduction observed at the concentrations of 0.98, 3.9 and 9.5 μg/L. Following a worst-case scenario, the NOEC for body weight was set at 3.9 μg/L based on the significant reduction at 9.5 μg/L. No statistically significant difference on body length was observed at any of the concentrations tested when compared to the pooled control.

Other effects:
- Only a few incidences of developmental defects were observed in the control groups and the test concentrations during the exposure period. The severity and incidence of abnormalities was not dose related.

Test medium:
- pH, dissolved oxygen, temperature and hardness were within the range described in the study plan

Reported statistics and error estimates:

The following statistical procedures were used to determine the NOEC/ECx values for embryonic and larval survival, body length and weight:

Comparison of control treatments:
• Hatchability and Larval survival: Fisher`s Exact Binomial Test (alpha=0.05, two-sided)
• Weight and Length: STUDENT-t test for Homogeneous Variances (alpha=0.05, twosided) after check for normality of distribution and homogeneity of variances.
As no statistically significant differences were detected for all parameters, the controls were pooled.

Hatchability (embryonic survival):
• ECx values could not be calculated as ≥96% embryos hatched in all treatments.
• Differences between treatments and the control: Chi²-2 x 2 table test with Bonferroni Correction (α=0.05, one-sided greater) after qualitative trend analysis by contrasts (monotonicity of concentration/response).

Larval survival:
• ECx-values could not be calculated as ≥92% of hatched larvae survived in all treatments.
• Chi2 2x2 Table Test with Bonferroni Correction (α=0.05, one-sided greater) after qualitative trend analysis by contrasts (monotonicity of concentration/response).

Larval growth (body length and weight):
• Due to the lack of a clear dose-response, no reliable ECx-values could be determined for body weight.
• No ECx-values for body length could be determined because the observed effects were below 10%.
• Body weight and length: Step-down Jonckheere-Terpstra Test Procedure (α=0.05, onesided smaller) after non-parametric trend analysis by contrasts (monotonicity of concentration/response). This statistical comparison was preceded by a check on normal distribution of the data (Shapiro-Wilk’s Test) and a test for homogeneity of the variances (Levene’s test).

All analyses were performed with ToxRat Professional 3.2.1 (ToxRat Solutions® GmbH, Germany)

Target and mean measured concentrations

Nominal conc. (µg/L)	Mean measured concentration (µg/L) at day					Mean exposure conc. (µg/L)	% Coefficient of variation
	0	7	14	21	28
0.30	0.334	0.173	0.683	0.269	0.326	0.36	54
0.94	0.776	0.592	1.18	0.889	1.47	0.98	35
3.0	2.98	3.05	3.66	2.43	7.37	3.9	51
9.4	6.16	11.4	12.6	7.55	9.84	9.5	28
30	28.4	37.7	42.0	27.7	41.8	36	20

 

Percentage of embryonic survival at the end of hatching (day 6)

Mean measured conc. (µg/L)	Total introduced	Hatched	Not hatched	% hatched
pooled control	160	160	0	100
0.36	80	77	3	96
0.98	80	78	2	98
3.9	80	80	0	100
9.5	80	80	0	100
36	80	79	1	99

 

Post-hatch mortality at the end of exposure (day 32)

Mean measured conc. (µg/L)	Total introduced (total number of hatched larvae)	Survived	Dead	% post-hatch mortality
pooled control	160	155	5	3.1
0.36	77	75	2	2.6
0.98	78	77	1	1.3
3.9	80	76	4	5.0
9.5	80	78	2	2.5
36	79	73	6	7.6

 

Mean fresh weight after 32 days of exposure

Mean measured conc. (µg/L)	Mean (mg)	Std. Dev.	n	% change
pooled control	55.787	3.6463	8
0.36	57.375	6.7653	4	2.9
0.98	53.075	1.5086	4	-4.9
3.9	53.200	1.6872	4	-4.6
9.5	50.050	1.9757	4	-10*
36	53.600	1.1690	4	-3.9*

* effect was statistically significant when compared to the pooled control

 

Mean body length after 32 days of exposure

Mean measured conc. (µg/L)	Mean (mm)	Std. Dev.	n	% change
pooled control	18.53	0.231	8
0.36	19.00	0.606	4	2.6
0.98	18.32	0.443	4	-1.1
3.9	18.58	0.189	4	0.27
9.5	18.25	0.451	4	-1.5
36	18.78	0.275	4	1.4

Validity criteria fulfilled:

yes

Remarks:

dissolved oxygen concentration above 60% throughout test, water temperature did not differ by more than 1.5°C, exposure concentrations were analytically monitored, overall survival of fertilised eggs in controls > 70% until hatching was complete

Conclusions:

The present study assessed the possible lethal and sub-lethal effects of JNJ42808415-AAA (T003422) during the embryonic and early larval development of the fathead minnow in a flow-through system (OECD 210). The early-life stages of fathead minnow were exposed to a range of concentrations of the test item dissolved in water until 32 days post fertilization. Lethal and sub-lethal effects were assessed and compared with control values to determine the various effect concentrations. The overall NOEC of JNJ-42808415-AAA (T003422) for the early life stages of fish was 3.9 µg/L and the overall LOEC was 9.5 µg/L. The results of the test can be considered reliable without restriction.

Description of key information

One key study (Augusiak, 2022) is used for endpoint coverage. The study investigates the chronic toxicity of T003422 to fish (Pimephales promelas) in a 32-day early-life stage toxicity test according to the OECD guideline 210. The study was carried out as a flow-through test, at nominal concentrations ranging from 0.30 to 30 µg/L, and using acetone as solvent. A 32d-NOEC of 3.9 µg/L was determined based on growth (mean measured concentration). The study was assigned a Klimisch score of 1 and was conducted under GLP.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

NOEC

Effect concentration:

3.9 µg/L