Climbazole

Endocrine disrupter testing in aquatic vertebrates – in vivo

Administrative data

Endpoint:

fish juvenile: (sub)lethal effects

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 November 2020 to March 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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 234 (Fish Sexual Development Test)

Version / remarks:

28th July 2011

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Climbazole

EC Number:

253-775-4

EC Name:

Climbazole

Cas Number:

38083-17-9

Molecular formula:

C15H17ClN2O2

IUPAC Name:

1-(4-chlorophenoxy)-1-(1H-imidazol-1-yl)-3,3-dimethylbutan-2-one

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

For the range finding test, sample analysis was conducted on at least one occasion prior Day 0 to confirm correct dosing. Samples were taken on Day 0 and at least on further occasion during the test.

For the definite test, samples (8 mL) of test media from the control and each test concentration were taken for analysis weekly throughout the duration of the test. A single replicate was sampled on each occasion with alternate replicates sampled each time with the exception of Day 0 (start of exposure) and the end of the test when each replicate was sampled.

Test solutions

Vehicle:

no

Details on test solutions:

An initial range-finding test was conducted at nominal concentrations of 0.0050, 0.050, 0.50 and 5.0 mg/L. Ten viable fish eggs were added to the test concentrations each and egg mortality, hatching success and early post-hatch survival was recorded after test duration of 10-14 days post-hatch.

Since the range finding study showed substance-related effects at a concentration of 0.50 mg/L, the concentration range for the definitive test was set as follows: 0.0050, 0.016, 0.050, 0.16 and 0.50 mg/L.

Test organisms

Aquatic vertebrate type:

fish

Test organisms (species):

Oryzias latipes

Details on test organisms:

TEST ORGANISM
- Common name: Japanese Medaka
- Source: Embryos were from breeding of adults from an in-house culture.
- Age at study initiation: eggs <12 hours old
- Brood stock selection: Apparently healthy looking parental fish were used for the breeding. Parental fish did not receive treatment for disease in the two-week period preceding breeding. Moribund fish or fish with clinical signs of disease were not used to produce the embryos used in the study.

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Numbers of parental fish: three breeding pairs
- Subsequent handling of eggs: eggs were gentely rolled to remove filaments, transfered into egg chambers
- Subsequent handling of juvenile fish: fish were gently released from egg chambers into the surrounding test media

FEEDING DURING TEST
- Food type: freshly hatched larvae will be fed ad libitum brine shrimp nauplii, later they will be fed ad libiturm old brine shrimp nauplii and/or Tetramin flake food
- Amount: ad libitum
- Frequency: 1-3 times daily
- Uneaten food and faecal material were removed from the test vessels daily 30 minutes after the last feeding by carefully cleaning the bottom of each tank using suction. Feeding was stopped 24 h before end of the test.

Study design

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

60 d

Test conditions

Hardness:

Control: 40-86 mg/L as CaCO3
Test solution: 52-87 mg/L as CaCO3

Test temperature:

25 ± 2 °C

Dissolved oxygen:

Control: 94.5-104.6 % of ASV (air saturation value);
Test solution: 92.8-103.4% of ASV (air saturation value)

Conductivity:

Control: 178-258 µS/cm
Test solution: 177-237 µS/cm

Nominal and measured concentrations:

Range finder: nominal 0.0050, 0.050, 0.50 and 5.0 mg/L. Measured 0.0055, 0.045, 0.45, 4.42 mg/L.


Definitive test: nominal 0.0050, 0.016, 0.050, 0.16 and 0.50 mg/L. Measured not yet anaylszed since study is still ongoing during the submission.

Details on test conditions:

TEST SYSTEM
- Test vessel: glass aquarium with egg chambers
- Material, size: Glass, the size of the test vessel was increased periodically as the size of the fish increased. Initially the test vessels contained ca 3 L of media per vessel which was then increased to ca 7 L per vessel.
- Type of flow-through: peristaltic flow-through system
- Renewal rate of test solution: the flow of the test media to the test vessels was controlled to give a flow rate of 12.5 ± 10% mL/minute.
- No. of organisms per vessel: 30 eggs
- No. of vessels per concentration: 4
- No. of vessels per control: 4
- Vehicle control performed: no
- Biomass loading rate: not exceeding 0.5 g of fish per litre in a 24-hour period.

TEST MEDIUM / WATER PARAMETERS
- Dilution water

OTHER TEST CONDITIONS
- Adjustment of pH: not necessary
- Photoperiod: 16-hour day: 8-hour dark
- Light intensity: 540 – 1000 lux

EFFECT PARAMETERS MEASURED :
- Survival – Daily assessment of survival will be made. Test embryos, larvae, and juvenile fish will be examined daily during the test period and any external abnormalities (such as haemorrhage, discoloration) noted. Any mortality will be recorded, and the dead organisms will be removed as soon as possible.

- Juvenil growth – The total length and wet weight of each fish will be determined and recorded at 30 days post-hatch. For wet weights, the fish will be placed into a vessel containing a small amount of water which will be pre-tarred prior the addition of the fish. The water will be carefully removed till a small amount still remains, but the fish is partially immobilized. The length of the fish will then be measured after which the fish will be returned to the test vessel.

- General observation – Abnormal behaviour (relative to controls) such as territorial aggressiveness, change of body colour etc. during the daily observations will be reviewed.
At test termination (approximately 60 days post-hatch) fish will be humanely euthanized. Total length and wet weight of each fish will be determined and recorded.

Sex characterization - The phenotypic sex of each fish will be identified based on dorsal and anal fin shape and secondary sex characteristics. Macroscopic examination of the gonads will also be conducted. If present, anal fin papillae will be counted.
The genetic sex of each fish will be determined. Therefore a small fin clip will be taken form the dorsal or ventral tip of the caudal fin of each fish. The DNA from the tissue sample will be extracted and the presence of absence of the gene dmy will then be determined by PCR method. The presence of dmy on the Y chromosome indicates a XY individual whilst the absence of dmy indicates a XX individual, regardless of phenotype.

- VTG – The measurement of VTG in plasma samples will be performed using ELISA. Therefore, the liver of each fish will be dissected for VTG analysis. If VTG analysis is not conducted immediately after sampling, the liver will be stored deep frozen until analysis is conducted. Where possible, at least 16 fish per replicate will be analyzed.

- Gonad sex (histopathology) – Whole fish will than be placed in fixative overnight, prior to storage in 10% neutral buffered formalin, for histological processing and microscopic examination. The samples will be stored at room temperature prior analysis.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2

RANGE FINDING STUDY
- Test concentrations: 0.0050, 0.050, 0.50 and 5.0 mg/L.
- Results used to determine the conditions for the definitive study: at a concentration of 0.50 mg/L the mortality is affected and at 5 mg/L the hatching success is 0% (please refer to the results section).

DATA ANALYSIS AND STATISTICS
Statistical analysis will be conducted using the CETIS program v 1.8.6.8.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

60 d

Dose descriptor:

NOEC

Effect conc.:

0.016 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

vitellogenin level

Effect concentrations 2

Duration:

60 d

Dose descriptor:

NOEC

Effect conc.:

0.05 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

sex ratio

Effect concentrations 3

Duration:

60 d

Dose descriptor:

NOEC

Effect conc.:

0.05 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

secondary sexual characteristics

Details on results:

See tables below

Any other information on results incl. tables

Table 1: Vitellogenin (VTG) analysis:

Nominal concentration (mg/L)	VTG concentration (ng/mL)
	Female Fish		Male Fish
	Mean	SD	Mean	SD
Control	1.56 x 10^5	0.15 x 10^5	29.90	56.84
0.0050	1.52 x 10^5	0.27 x 10^5	12.55	16.42
0.016	1.82 x 10^5	1.15 x 10^5	81.45	156.51
0.050	0.71 x 10^5	0.14 x 10^5	19.41	38.50

Table 2: Sex ratio:

Nominal concentration (mg/L)	Genetic sex		Phenotypic sex
	Proportion of total number (%)		Proportion of total number (%)
	Female fish	Male fish	Female fish	Male fish
Control	56	44	55	45
0.0050	49	51	50	50
0.016	48	52	49	51
0.050	43	58	50	50

Table 3: Secondary Sex Characteristics:

Nominal concentration (mg/L)	Number of anal fin papillae
	Mean	SD
Control	66	5.0
0.0050	63	6.7
0.016	66	6.1
0.050	71	22.6

 

Table 4: Histological evaluations:

Nominal concentration (mg/L)	Number of individuals
	XX male	XY female	Intersex
Control	1	-	-
0.0050	-	1	-
0.016	-	1	1
0.050	1	1	1

- = not applicable

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Based on the observed results, it is considered that the test item does not show any adverse endocrine effects at the concentrations tested.

Executive summary:

For evaluation the potential endocrine effects of the substance a 60-day fish sexual development study on medaka (Oryzias latipes) under flow-through conditions was conducted according to OECD TG 234 and GLP. Based on the results from a range finding test, the concentration range for the definitive test was selected as follows: 0.0050, 0.016, 0.050, 0.16 and 0.50 mg/L. The substance concentrations were analysed via LC-MS/MS and maintained between 80 and 120% of nominal concentrations.  At the end of the exposure, the adult fish were assessed for growth, secondary sex characteristics, genetic sex and vitellogenin (VTG). Gonad histology was performed to determine the phenotypic sex of the fish. In addition, gonad, liver and kidney histopathology was performed. The observed results show that Sex ratio did not appear to be affected by the test substance at the concentrations employed with no significant differences observed between the test concentrations and combined controls in terms of either genetic or phenotypic sex ratio; No statistically significant increase in VTG concentration in male fish was observed and no statistically significant effects were observed for male secondary sex characteristics. Inconclusion, it is considered that the test item does not show any adverse endocrine effects at the concentrations tested.