1,3-dihydro-4(or 5)-methyl-2H-benzimidazole-2-thione

Endocrine disrupter testing in aquatic vertebrates – in vivo

Administrative data

Endpoint:

amphibian Xenopus laevis, larvae: (sub)lethal effects

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 April 2021 - 20 September 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This information is submitted based on ECHA decision number SEV-D-2114512626-52-01/F

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD TG 231 (The Amphibian Metamorphosis Assay)

Version / remarks:

2009

Deviations:

yes

Remarks:

None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions. See Appendix 7 of the attached full study report for further details.

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 10-17 December 2019 / Date of signature: 24 February 2020

Test material

Test material information

Constituent 1

Reference substance name:

1,3-dihydro-4(or 5)-methyl-2H-benzimidazole-2-thione

EC Number:

258-904-8

EC Name:

1,3-dihydro-4(or 5)-methyl-2H-benzimidazole-2-thione

Cas Number:

53988-10-6

Molecular formula:

C8H8N2S

IUPAC Name:

1,3-dihydro-4(or 5)-methyl-2H-benzimidazole-2-thione

Test material form:

solid: particulate/powder

Details on test material:

- Physical description: white powder
- Storage conditions: at room temperature
- Volatile: no
- Solubility in water: 0.12 g/L
- Stability in water: stable

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Samples were taken from all test concentrations and the control.
- Frequency: One day before the start of exposure to check the functioning of the system in one replicate of each group. At the start (Day 0, in the morning and late afternoon) and after 1, 7, 14 and 21 days of exposure from all replicate test solution of each test group. Stock solutions were sampled on Day 0 (in the morning and late afternoon), 1, 7, 14 and 21.
- Sampling method: Volume 1.8 mL (Day -1, 0, 1) or 3.5 mL (Day 7, 14, 21).
- Sample storage conditions before analysis: Samples were transferred to the analytical laboratory at the Test Facility and analyzed on the day of sampling, except for Day 0 late afternoon samples, which were stored in a freezer (set to maintain -20°C) until analysis at the analytical laboratory of the Test Facility.

Additionally, reserve samples of 1.8 mL (Day 0, 1) or 3.5 mL (Day 7, 14, 21) were taken from all stock and test solutions for additional analysis if needed. If not already used, these samples were stored in a freezer (set to maintain -20°C) for a maximum of three months after the end of the test.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stock solutions for dosing were prepared every two days. Preparation of stock solutions started with a loading rate of 100 mg/L applying a one-day period of continuous stirring to ensure maximum dissolution of the test item in test medium. Thereafter, the aqueous Saturated Solution (SS) was collected by filtration through a 0.45 µm membrane filter (RC55, Whatman). The stock solutions were dosed to test medium by means of a computer-controlled dosing system. For the four lowest concentrations, exact volumes of the test item stock solutions were dosed with syringes via a computer-controlled system consisting of four dispensers (Gilson). For the two highest test concentration, exact volumes of the test item stock solutions were dosed using peristaltic pumps. The dosed volumes of the stock entered a mixing flask separately from the exposure medium supply. The dilution water was supplied applying flowmeters at a constant rate of 8 L per hour. In the mixing flask, the dosed volume and the exposure medium were mixed under continuous stirring. A peristaltic pump unit transported the test solutions from the mixing vessel to the replicates of each test concentration at a rate of 1.5 L per hour per test vessel. In addition, a stock solution of KI was supplied to the mixing flask at a constant rate resulting in a concentration of 3.27 µg KI/L. Performance of the dosing system was checked daily. Table 6.1.8/1 in "Any other information on materials and methods incl. tables" shows details on the dosing used during the 21-day exposure period test. Any residual volumes were discarded.
- Controls: Test medium without test item or other additives.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable.
- Test concentration separation factor: 5
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): All stock solutions were clear and colorless at the end of the preparation procedure.

Test organisms

Aquatic vertebrate type:

frog

Test organisms (species):

Xenopus laevis

Details on test organisms:

TEST ORGANISM
- Common name: Xenopus laevis (Daudin), class: Amphibia, order: Anura, family: Pipidae.
- Source: One-day-old eggs were purchased from the European Xenopus Resource Centre, University of Portsmouth, UK. Further embryos and tadpoles were cultured at Charles River Den Bosch until stage 51.
- Life stage: 13 days post fertilization.
- Age at study initiation (mean and range, SD): Normally developed tadpoles of developmental (NF) stage 51.
- Length at study initiation (length definition, mean, range and SD): Tadpoles were between 28 and 32 mm long (average 31 mm) at test commencement.
- Weight at study initiation (mean and range, SD): no data
- Method of breeding: See "REARING" details below.

REARING
- Larval care and selection: The three best individual spawns were retained based upon embryo viability and the presence of an adequate number of embryos. All the organisms used in the study originate from a single spawning event (i.e., the spawns were not co-mixed). The sound embryos from each of the three spawns were transferred into three separate hatching tanks. Four days after being placed in the hatching tanks, the best spawn, based on viability and hatching success, was selected and the larvae were transferred into a rearing tank. The rearing tank was siphoned clean daily. Mortalities were removed daily.
- Medium: Test medium
- Flow rate: 50 mL/min
- Temperature: 22 ± 1°C
- Illumination: A daily photoperiod of 12 hours with an intensity between 600 and 2000 lux.
- Age of the tadpoles: From one day after fertilization to NF stage 51
- Rearing tank:
Day 0 – 4: at least 20 litre all glass aquaria covered by a removable Perspex plate.
Day 4 onwards: 100 litres (50x50x40 cm) consisting of stainless steel and covered by a removable Perspex plate (rearing density was kept at ≤10 tadpoles/L medium applying a flow rate of approximately 50 mL/min).
- Feeding: From stage 44/45 with Sera Micron® (Sera GmbH, Heinsberg, Germany) at least twice per day.
- Aeration: Continuous

FEEDING DURING TEST
At least twice daily with Sera Micron® (Sera GmbH, Heinsberg, Germany) according to the scheme given in the Table 6.1.8/2 in "Any other information on materials and methods incl. tables". At Day 17 of exposure a number of tadpoles reached NF stage 61 at which they do not forage. Therefore, the amount of food supplied was accordingly reduced, i.e. was supplied in amounts needed to feed the tadpoles that were in NF stage 60 or below.

Study design

Test type:

flow-through

Water media type:

freshwater

Remarks:

Salts added for reconstitution of the reverse osmosis (RO) water. See "details on test conditions" for further information.

Limit test:

no

Total exposure duration:

21 d

Test conditions

Hardness:

214-250 mg/L expressed as CaCO3.

Test temperature:

Measured separately in every tank at weekly intervals: 22.0-23.4°C.
Measured continuously in control tank: 21.0-22.6°C.

pH:

7.2-7.7

Dissolved oxygen:

4.8-8.5 mg/L

Conductivity:

592-601 µS/cm

Nominal and measured concentrations:

- Nominal concentrations: 0.0048, 0.024, 0.12, 0.60, 3.0 and 15 mg/L.
- Mean measured concentrations: 0.0052, 0.027, 0.15, 0.72, 3.3 and 16 mg/L. See Table 6.1.8/3 in "Any other information on results incl. tables".

Details on test conditions:

TEST SYSTEM
- Test vessel: Approximately 10 L (42 x 13.5 x 19.5 cm (LxWxH)) consisting of glass plates sealed with a thin film of silicone and covered by a removable Perspex plate.
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: Each vessel contained approximately 10 L of medium (water depth of ~16 cm).
- Aeration: Aeration was continuous during the exposure.
- Type of flow-through (e.g. peristaltic or proportional diluter): peristaltic
- Renewal rate of test solution (frequency/flow rate): Flow rate 25 mL/min
- No. of organisms per vessel: 20
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Vehicle control performed: not applicable
- No. of vessels per vehicle control (replicates): not applicable
- Biomass loading rate: The experiment (nominal Day 0) started with 80 tadpoles of NF stage 51, per concentration and the control. The tadpoles were randomly distributed and divided equally over four replicates per treatment (20 per replicate).
- Terminal procedures - Euthanasia: On Day 0 (tadpoles not used for the test), Day 7 (5 tadpoles/replicate) and at the end of the test all remaining tadpoles were rapidly killed by exposing them to ca. 1.2% ethylene glycol monophenylether in water.

TEST MEDIUM / WATER PARAMETERS
The following salts (analytical grade) were added to tap water purified by reverse osmosis (RO-water, GEON Waterbehandeling, Berkel-Enschot, The Netherlands):
CaCl2.2H2O 211.5 mg/L
MgSO4.7H2O 88.8 mg/L
NaHCO3 46.7 mg/L
KCl 4.2 mg/L
KI 3.27 µg/L
Hardness of 180 mg/L expressed as CaCO3.

OTHER TEST CONDITIONS
- Adjustment of pH: no.
- Photoperiod: daily photoperiod of 12 hours.
- Light intensity: 792-795 lux (start exposure); 691-695 lux (end exposure).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Mortality and abnormal appearance/behavior: Observations of mortality and abnormal appearance/ behavior were made daily. Abnormal behaviour can include floating on the surface, lying on the bottom of the tank, inverted or irregular swimming, lack of surfacing activity, and being non-responsive to stimulus. In addition, gross differences in food consumption between treatments can be considered as abnormal behaviour. Gross malformations and lesions could include morphological abnormalities (e.g., limb deformities), haemorrhagic lesions and/or bacterial or fungal infections. These determinations are qualitative and should be considered akin to clinical signs of disease/stress and made in comparison to control animals. In case the occurrence or rate of occurrence were greater in exposed tanks than in the controls, then these were considered as evidence for overt toxicity. Similar lesions manifested across all groups, including the control group, should be considered as evidence of inappropriate testing conditions.
Criteria for death: Immobility and/or absence of respiratory movement and/or lack of reaction to mechanical stimulus. Dead tadpoles were removed when observed.
- Development (NF) stage, Hind Limb Length (HLL), Snout-to-Vent Length (SVL) and Wet Weight (WW): Day 0 (only development stage). Day 7 on a subsample of 5 randomly chosen tadpoles per replicate. Day 21 for all remaining tadpoles. On both occasions (Day 7 and 21), all measurements were taken on previously euthanized animals. On day 7 and 21, photos of euthanized tadpoles were taken and SVLs and HLLs were measured using AxioVision software (v. 4.8.).
- Thyroid Histopathology: Animals selected for histopathology (n=5 from each replicate) were matched to the median stage of the blank control (pooled replicates); i.e. stage 61 where possible. As there were not enough tadpoles of this stage in most of the replicates, tadpoles of stage 55 (only the highest concentration), 58 (only the second highest concentration), 59 (all concentrations) and 60 (all concentrations) were also included in order to provide 5 tadpoles per replicate. Collection: Euthanized tadpoles were staged, weighed, photographed and immediately thereafter immersed in modified Davidson’s fixative (1-2 minutes after euthanizing). After approximately 48-96 hours fixation, tadpoles were transferred to neutral buffered formalin (10%).

TOXICITY TEST / RANGE FINDING STUDY
Since no data was available regarding the toxicity of the test item to Xenopus laevis or other amphibians, a 14-day toxicity test in Xenopus laevis was performed as a concentration range-finding test for the definitive test.
- Test concentrations: 0.10, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: The measured concentrations in the test concentrations were at 87-97% of the nominal concentrations one day before and at the start of the exposure period confirming proper functioning of the dosing system. No mortality or remarkable adverse effects were observed in the control and the three lowest concentrations (0.10, 1.0 and 10 mg/L) during the 14-day test period. In the highest concentration (100 mg/L), all tadpoles died within 24 hours of exposure. Developmental stage at the end of exposure seemed to indicate a potential for retardation of development at 10 mg/L. Developmental stage ranged between 54 and 56 in all test groups. No statistically significant reduction was observed in wet body weight when comparing the test item treated groups to the control group. The 14-day LC50 for Xenopus laevis tadpoles with NF stage of 51 at test start was determined to be 32 mg/L under flow-through conditions. Based on the results of the toxicity test, and to meet the expectations of the ECHA Decision on Substance Evaluation Letter, the following concentrations were chosen for the full test: 0.0048, 0.024, 0.12, 0.60, 3.0 and 15 mg/L.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

developmental stage

Effect concentrations 2

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

wet body weight

Effect concentrations 3

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

4.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

wet body weight

Effect concentrations 4

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

snout-vent length

Remarks:

(SVL)

Effect concentrations 5

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

snout-vent length

Remarks:

(SVL)

Effect concentrations 6

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

hind limb length

Remarks:

(normalized by SVL)

Effect concentrations 7

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Effect concentrations 8

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

behaviour

Remarks:

/ Appearance

Effect concentrations 9

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

developmental stage

Effect concentrations 10

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

wet body weight

Remarks:

(NF stage ≤60)

Effect concentrations 11

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.024 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

wet body weight

Remarks:

(NF stage >60)

Effect concentrations 12

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

snout-vent length

Remarks:

(SVL) (NF stage ≤60)

Effect concentrations 13

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.024 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

snout-vent length

Remarks:

(SVL) (NF stage >60)

Effect concentrations 14

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.024 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

hind limb length

Remarks:

(normalized by SVL)

Effect concentrations 15

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.024 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

histopathology

Remarks:

(Thyroid Gland)

Details on results:

See tables in "Any other information on results incl. tables".
The performance criteria were used to determine the quality of the test and the general performance of the control organisms and are summarized in table 6.1.8/5 in "Overall remarks". The performance criteria described by the guideline were met.

MEASURED TEST ITEM CONCENTRATIONS
In the definitive test, the actual concentrations measured before test start were lower than expected based on the results of the range-finding test. Only about 70% of nominal were measured in the stock solutions. The filter material was the same as in the range-finding test, and the prepared volumes were comparable. It is thus not clear what caused the difference. The result of the stock from Day -1 was furthermore double-checked with the reserve sample and another sample from Day 0. However, concentrations were confirmed. Consequently, it was decided to adjust the dosing flow rates for the stock solution to get the test vessel concentrations as close to the target values as possible. Also, extra stock solution volumes were added to the test vessels to shorten the time that would otherwise be needed for concentrations to change in the test vessels. This was followed by extra sampling from all test vessels before the tadpoles were added. The next morning, additional samples were taken to determine the test vessel concentrations after a night with the adjusted flow rates. The Day 0 results indicated for most cases a successful increase of concentrations in the vessels, but at the lower levels, the contribution of added stock was not as evident. This might be related to not waiting long enough between adding the extra stock and taking the sample to allow homogeneous distribution of the added stock solution. As the tadpoles were added only once sampling was completed, it is assumed that sufficient time passed for this process to complete before exposure started. The Day 1 results subsequently indicated dosing close to the target levels. The following results confirm stable dosing and generally repeatable preparation of stock concentrations.
On Day 21, the concentration of the stock solution was 93 mg/L and as such higher than observed on previous occasions of the definitive test. Similar to the start of the test, no reason for this could be identified. The filter material was the same as used throughout the study, and the prepared volumes were comparable. The calculated mean exposure concentrations are presented in Table 6.1.8/3 in "Any other information on results incl. tables". The mean measured concentrations were at the level of 107 and 121% of the nominal concentrations. The coefficient of variation was between 19 and 27% for the five highest concentrations. For the lowest concentration, this was 43%. It should be noted that in some samples the measured concentrations were slightly below the LOQ of the analytical method, especially in the Day 0 samples, where the added amounts of stock solution may not have been completely homogenized throughout the test vessels at the time of sampling. Without the Day 0 samples, the coefficient of variation was determined to be 28% and thus more in line with the remaining test concentrations. In conclusion, the values for %CV were accepted as the technically best possible outcome considering that the stock solutions were prepared as saturated solutions. Overall dosing was considered sufficiently stable at graded levels to allow a reliable interpretation of results. Since the mean concentrations were generally within ±20% of the nominal concentration, with one exception being at 121% of nominal, the subsequent results were evaluated against the nominal concentrations.

MEASURED CONCENTRATIONS OF IODIDE IN WATER SAMPLES
The analysis of iodide in the samples taken from the control solutions showed an average concentration of 2.51 µg/L, which corresponded to 77% of the nominal concentration of 3.27 µg/L, and was within the recommended range of 0.5 to 10 µg/L.

SURVIVAL AND SYMPTOMS
Mortality and symptoms observed in this study are summarized in Table 6 of the attached full study report.
No mortality was observed in any of the replicates of the control group. Mortality in test item treated groups was limited to one specimen in the lowest concentration (1.3%) and to one specimen in the 0.12 mg/L group (1.3%). No concentration-response relationship could be observed, deaths were not considered test item related and none of the replicates were considered compromised. The NOEC for survival was thus set at 15 mg/L (LOEC ≥15 mg/L). In each replicate, a number of tadpoles with scoliosis was observed, except for in the highest concentration. One tadpole in the control group was missing the left hind leg. These observations were not considered to be concentration related as there was no clear relationship between concentration and the number of observations. Tadpoles exposed to 15 mg/L were generally less active compared to the control during the first half of exposure with single tadpoles in this concentration floating on their back during the first week of exposure. At the end of exposure, several tadpoles exposed to 15 mg/L had dark green to black colored spots on their heads. The observations in the highest concentration were not considered signs of overt toxicity as this did not appear to cause direct discomfort to the tadpoles.

DEVELOPMENT
Two endpoints indicative of metamorphosis, and as such sensitive to changes in the functioning of the HPT axis, are the NF developmental stage and hind limb length (normalized for snout-to-vent length). NF stages of individual tadpoles are presented in Table 7 (Day 7) and Table 9 (Day 21) of the attached full study report. Individual data on normalized HLL are presented in Table 8 (Day 7) and Table 10 (Day 21) of the attached full study report.
- NF developmental stage:
At Day 7, no statistically significant effects were found at any of the concentrations when compared to the control. Based on these results, no acceleration and/or retardation of development was indicated after 7 days of exposure.
At Day 21, the group median NF developmental stage was 61 in the control treatment and 61, 62, 62, 61, 59 and 55 at concentrations of 0.0048, 0.024, 0.12, 0.60, 3.0 and 15 mg/L, respectively. The NF developmental stage after 21 days of exposure was significantly lower in the 15 mg/L concentration compared to the control. This indicates a retardation of development after 21 days of exposure in the highest concentration. No cases of accelerated or asynchronous development were observed in this study.
- Normalized Hind Limb Length:
At Day 7, no statistically significant effects were found at any of the concentrations when compared to the control treatment, except for the 3.0 mg/L concentration, where the normalized HLL was significantly lower compared to the control. Due to a lack of concentration response, this finding was not considered as a test item related effect. Based on these results, no acceleration and/or retardation of development was indicated after 7 days of exposure.
After 21 days of exposure, the mean normalized hind limb length was statistically significantly higher in the 0.12 mg/L concentration and was found to be statistically significantly lower in the 3.0 mg/L and 15 mg/L concentrations compared to the control. These findings indicate a delay of development in the two highest concentrations. These results are consistent with the observations of retarded development and suggest that the test item affects metamorphosis of Xenopus laevis after 21-day exposure to different concentrations of this test item.

GROWTH
Effects on growth were measured as changes in wet weight (WW) and Snout-to-Vent length (SVL). It should be noted that effects on Hind Limb Length (HLL) are not used as standalone data for evaluation of toxic or endocrine effects. The HLL normalized for SVL is used as an indicator of endocrine disrupting effects.
Since at least 20% of tadpoles in all but two concentrations of the test item showed development beyond NF stage 60, the analysis of data on SVL and WW was performed after grouping individual tadpoles according to the stage, i.e. LateStage was defined as “No” if tadpoles were in NF stage ≤60 and as “Yes” if in NF stage ≥61. This was necessary as after NF stage 60, natural decrease of body weight and resorption of tail occurs, which confounds results.
- Wet weight:
The effects of the test item on WW at Day 7 is presented in Table 11 of the attached full study report. The mean wet body weight on Day 7 was significantly lower for the 15 mg/L concentration compared to the control. No statistically significant effects were observed in other concentrations. The NOEC was thus set at 3.0 mg/L. The LOEC was accordingly set at 15 mg/L. No suitable regression model could be identified to determine ECx-values. The EC10- and EC20-values were accordingly estimated by linear interpolation using the information of the control and the highest test concentration. The EC10 was determined to be 4.4 mg/L and the EC20 was determined to be 8.7 mg/L.
The effects of the test item on WW at day 21 is presented in Table 13 of the attached full study report. The mean wet body weight for tadpoles with NF stage ≤60 was statistically significantly higher in the 3.0 mg/L concentration compared to the control and statistically significantly lower in the 15 mg/L concentration compared to the control. The NOEC was thus set at 0.60 mg/L. The LOEC was accordingly set at 3.0 mg/L for tadpoles of NF stage ≤60. No ECx-values could be determined reliably for tadpoles of NF stage ≤60 as the observed effects did not follow a clear concentration-response pattern. For tadpoles with NF stage ≥61, the mean wet body weight was statistically significantly lower in the 0.12 mg/L concentration compared to the control. The observed changes of wet weight in test item treated groups did not follow a clear concentration-response curve. Following a worst-case assumption approach, the NOEC for tadpoles of NF stage ≥61 was set at 0.024 mg/L despite the higher concentrations not showing statistically significant difference from the control group. The LOEC was accordingly set at 0.12 mg/L for tadpoles of NF stage ≥61. No ECx-values could be determined reliably for tadpoles of NF stage ≥61 as the observed effects did not follow a clear concentration-response pattern.
- Snout-to-Vent Length:
The effects of the test item on SVL at day 7 is presented in Table 12 of the attached full study report. The mean SVL on Day 7 was significantly lower in the 15 mg/L concentration compared to the control. No statistically significant effects were observed in other concentrations. The NOEC was thus set at 3.0 mg/L. The LOEC was accordingly set at 15 mg/L. No suitable regression model could be identified to determine ECx-values. The EC10-value was consequently estimated by linear interpolation using the information of the control and the highest test concentration. The EC10 was determined to be 10 mg/L. Considering that no clear retardation was observed in the developmental parameters, these findings on WW and SVL at day 7 likely suggest systemic toxic effects rather than anti-thyroidal mechanisms.
The effects of the test item on SVL at day 21 is presented in Table 14 of the attached full study report. The mean SVL within tadpoles with NF stage ≤60 was significantly higher in the 3.0 mg/L concentration compared to the control. The observed changes of SVL in test item treated groups did not follow a clear concentration-response curve. Following a worst-case assumption approach, the NOEC for tadpoles of NF stage ≤60 was set at 0.60 mg/L despite the higher concentration not showing a statistically significant difference from the control group. The LOEC was accordingly set at 3.0 mg/L. No ECx-values were determined as the observed effects were below 10%. Within tadpoles with NF stage ≥61, the mean SVL was significantly lower in the 0.12 mg/L concentration compared to the control. The observed changes of SVL in test item treated groups did not follow a clear concentration-response curve. Following a worst-case assumption approach, the NOEC for tadpoles of NF stage ≥61was set at 0.024 mg/L despite the higher concentration not showing a statistically significant difference from the control group. The LOEC was accordingly set at 0.12 mg/L. No ECx-values were determined as the observed effects were below 10%.
Growth parameters are investigated to detect systemic toxicity of a test item. Thus, the obtained results indicate that an appropriate range of concentrations was tested in the present study as the highest group showed slight toxic, but not life-threatening effects as required by the OECD Technical Guidance 150 (OECD, 2018).

HISTOPATHOLOGICAL OBSERVATION
At day 21, five tadpoles per test group were processed for histopathological evaluation of the thyroid gland. Test item-related microscopic alterations in the thyroid gland were observed starting at a concentration of 0.12 mg/L. These findings consisted of thyroid hypertrophy (starting at 0.60 mg/L), follicular cell hypertrophy (starting at 0.12 mg/L), dilatation of follicles (starting at 0.60 mg/L) and follicular cell hyperplasia (starting at 0.60 mg/L), with all parameters designated as severe degree at doses of 3.0 and 15 mg/L. Based on the thyroid histopathology, the test item would be classified as a thyroid-active substance.
Details on method and results are given in Appendix 6 of the attached full study report.

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

See Appendix 3 of the attached full study report.

Any other information on results incl. tables

Table 6.1.8/3: Nominal and mean measured concentrations

Test item Nominal conc. (mg/L)	Repl.	Mean measured concentration (mg/L) at day					Group mean (mg/L)	%CV^1	Relative to target [%]
		0	1	7	14	21
0.0048	A	0.01076	0.00505	0.00654	0.00380^+	0.00523	0.0052	43	108
	B	0.00212^+	0.00496	0.00630	0.00382^+	0.00975
	C	0.00223^+	0.00494	0.00635	0.00386^+	0.00505
	D	0.00201^+	0.00499	0.00638	0.00388^+	0.00533
0.024	A	0.0142	0.0270	0.0348	0.0206	0.0338	0.027	26	113
	B	0.0141	0.0257	0.0348	0.0203	0.0341
	C	0.0269	0.0336	0.0340	0.0207	0.0336
	D	0.0209	0.0267	0.0342	0.0205	0.0326
0.12	A	0.133	0.133	0.173	0.105	0.182	0.15	19	121
	B	0.136	0.134	0.172	0.104	0.176
	C	0.132	0.136	0.173	0.104	0.176
	D	0.165	0.131	0.169	0.105	0.178
0.60	A	0.776	0.653	0.851	0.517	0.851	0.72	20	119
	B	0.652	0.662	0.858	0.511	0.876
	C	0.707	0.669	0.858	0.512	0.893
	D	0.538	0.668	0.866	0.513	0.883
3.0	A	3.40	3.02	3.81	2.35	3.94	3.3	19	110
	B	3.20	2.90	3.87	2.29	4.00
	C	3.63	2.93	3.93	2.37	3.85
	D	3.66	2.96	3.81	2.28	3.92
15	A	16.2	13.2	17.1	10.7	23.1	16	27	107
	B	16.5	13.4	17.0	11.3	22.7
	C	14.9	13.3	16.8	10.6	23.5
	D	16.0	12.9	17.2	10.8	22.7
100	Stock	67.9	69.7	79.7	62.6	92.6	74	16	74

¹ Coefficient of variation; ⁺ Analyzed concentration is below limit of quantification (LOQ) of 0.0045 mg/L.

 

Table 6.1.8/4: Summary of effect parameters measured

Apical Endpoints		Test item, Nominal conc. (mg/L)
		0.0048	0.024	0.12	0.60	3.0	15
Day 7	Developmental Stage	-	-	-	-	-	o
	Wet Weight ^1	-	-	-	-	o	↓
	Snout-Vent Length (SVL) ^2	-	-	-	-	o	↓
	Hind Limb Length (normalized by SVL)	-	-	-	o	↓	-
Day 21	Survival	-	-	-	-	-	o
	Behaviour / Appearance	-	-	-	-	o	+
	Developmental Stage	-	-	-	-	o	↓
	Wet Weight (NF stage ≤60) Wet Weight (NF stage >60)	- -	- o	- ↓	o -	↑ -	↓ n.d.
	Snout-Vent Length (SVL) (NF stage ≤60) Snout-Vent Length (SVL) (NF stage >60)	- -	- o	- ↓	o -	↑ -	- n.d.
	Hind Limb Length (normalized by SVL)	-	o	↑	-	↓	↓
	Thyroid Gland Histopathology	-	o	+	+	+	+
- Endpoint not impacted compared to the control (qualitative assessment) (qualitative assessment) ↓ Endpoint significantly reduced ↑ Endpoint significantly increased n.d. – Not determined. No suitable tadpoles available for assessment. o Identified NOEC ^1 EC10: 4.4 mg/L, EC20: 8.7 mg/L ^2 EC10: 10 mg/L

 

 

 

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study, the test item demonstrated an effect on metamorphosis (measured as NF developmental stage and normalized hind limb length) and histology of the thyroid glands of Xenopus laevis tadpoles during the exposure. Therefore, it was concluded that the test item has a potential to interfere with the hypothalamus-pituitary-thyroid axis of vertebrates. The lowest 21d-NOEC value was determined at 0.024 mg/L.

Executive summary:

This Amphibian Metamorphosis Assay (AMA) was performed to identify whether the test item may interfere with the normal function of the hypothalamic-pituitary-thyroid (HPT) axis, according to OECD Test Guideline 231 with GLP statement. The AMA represents a generalized vertebrate model to the extent that it is based on the conserved structures and functions of the HPT axis. The African clawed frog (Xenopus laevis) was used for this purpose.

According to a 14-days toxicity test, Xenopus laevis tadpoles were exposed to the test item at analytically confirmed nominal concentrations of 0.0048, 0.024, 0.12, 0.60, 3.0 and 15 mg/L and to a control, over a period of 21 days under flow-through conditions. Four replicates were used, each containing 20 tadpoles of Developmental (NF) stage 51 at the start of the test, except for one replicate of the control which had received 19 tadpoles of NF stage 51 and two replicates in the highest test concentration which had received, both, 21 tadpoles of NF stage 51.

The study met the acceptability criteria and was considered valid.

On Day 7 of exposure, effects observed at the two highest test concentrations suggest a slight delay to development coupled with overt signs of toxicity. As elaborated in the OECD TG 231, mild developmental delays coupled with overt signs of toxicity likely indicate a non-specific toxic effect. Similarly, mild reductions in growth, as determined by Wet Weight (WW) and/or Snout-to-Vent Length (SVL), also suggest non-thyroidal toxicity. Considering that no clear retardation was observed in the developmental parameters, the recorded findings on WW and SVL at Day 7 likely suggest systemic toxic effects rather than anti-thyroidal effects.

On Day 21 of exposure, several observations were made at the highest two test concentrations that were related to retardation of development. Firstly, the group median developmental stages were slightly to significantly lower than in the control; the difference of developmental stages was statistically significant at the highest concentration. Additionally, Hind Limb Length (HLL) normalized for SVL at the two highest test concentrations was significantly lower than in the control, suggesting a decreasing trend in the normalized HLL as concentration increased.

These two endpoints are associated with metamorphosis and as such, are sensitive to changes in the functioning of the HPT axis. As mentioned above, delayed development can occur through two ways - anti-thyroidal mechanisms or indirect toxicity. Mild developmental delays coupled with overt signs of toxicity likely indicate a non-specific toxic effect.

In the present study, no excessive mortality was observed but other signs of systemic toxicity were observed, i.e. SVL and wet body weight were clearly affected by test item exposure. On the other hand, developmental delays at the end of exposure could not be considered mild as there was no overlap of developmental stages in the highest test concentration and the control group. Also, the histopathological assessment of the thyroid glands indicated clear tissue alterations related to test item exposure.

It should be noted that the developmental delay at the higher concentrations conflicted with the possibility of selecting tadpoles matched to the median developmental stage in the control group. Following Grim et al. (2009), the differences in thyroid gland histopathology between NF stages 57 and 62 are not strong enough to conflict with the conclusions drawn by the histopathological assessment in the present study for the different treatment levels. Based on the observed thyroid histopathology, the test item clearly affected the thyroid gland of exposed tadpoles. The lowest 21d-NOEC value was determined at 0.024 mg/L. 

To conclude, neither advanced, nor asynchronous development were observed in the present study. However, remarkable histological effects were noted at concentrations of 0.12 mg/L and higher, including assignment of ‘severe degree’ grading for the core assessed criteria at 3.0 mg/L and 15 mg/L. Consequently, following the decision logic presented in OECD TG 231, the test item is concluded to be thyroid active. As no mortality occurred in the control group, the study is considered valid.