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EC number: 253-775-4 | CAS number: 38083-17-9
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
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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:
- 2 021
- 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
- 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
Constituent 1
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 concentrationsopen allclose all
- 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
- 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
- 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 | VTG concentration (ng/mL) | |||
Female Fish | Male Fish | |||
Mean | SD | Mean | SD | |
Control | 1.56 x 105 | 0.15 x 105 | 29.90 | 56.84 |
0.0050 | 1.52 x 105 | 0.27 x 105 | 12.55 | 16.42 |
0.016 | 1.82 x 105 | 1.15 x 105 | 81.45 | 156.51 |
0.050 | 0.71 x 105 | 0.14 x 105 | 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 | 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.
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