Registration Dossier

Administrative data

Endpoint:
fish, juvenile growth test
Remarks:
In vivo experimental information for endocrine activity discussion.
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Vitellogenin analysis not sex or organ specific
Justification for type of information:
Experimental information for endocrine activity discussion.

Data source

Reference
Reference Type:
publication
Title:
Comparison of in vitro and in vivo estrogenic activity of UV filters in fish
Author:
Kunz PY, Galicia HF, Fent K
Year:
2006
Bibliographic source:
Toxicological Sciences 90(2): 349-361

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
14 day exposure of juvenile fathead minnow (Pimephales promelas). Endpoints: length, weight and estrogenic activity (vitellogenin induction)
GLP compliance:
not specified
Remarks:
Published study from a peer-reviewed journal.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Obtained from Merck (Glattbrugg, Switzerland) >99% purity.

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
At each renewal, exposure water was taken from the two highest and the two lowest concentrations of the test substance and controls at the beginning (0 h) and prior to water renewal (24 h). They were preserved by acidification using HCl to pH 2–3, and stored at 4oC until analysis

Test solutions

Vehicle:
yes
Remarks:
1 ml ethanol in 10 liters of water

Test organisms

Test organisms (species):
Pimephales promelas
Details on test organisms:
TEST ORGANISM
- Age at start of exposure: Juvenile sexually undifferentiated fathead minnows (Pimephales promelas), between 2 and 3 months of age and with a total body length between 19 and 27 mm.
- Feeding during test: Fish were fed with brine shrimp (Artemia salina, Argent Chemical Labs, Redmond WA, USA) at a feeding rate of 1% of body weight twice a day.

SOURCE OF FISH: Mixed-sex juvenile fathead minnows were received from Aquatic Research Organisms, Hampton NH, USA) and adapted for a minimum of 14 days in the laboratory in aquaria prior to the experiment.

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
14 d

Test conditions

Hardness:
Total hardness 160 mg/l as CaCO3
Test temperature:
25oC ± 1oC
pH:
pH ranged between 7.2–7.9
Dissolved oxygen:
Oxygen saturation ranged between 6.5–8.3 mg/l
Conductivity:
Conductivity 500 us/cm
Nominal and measured concentrations:
Solvent control and 10, 100, 500, and 1000 ug/l 4-methylbenzylidene camphor. Analytical verification of the nominal 10, 500 and 1000 µg/L exposures had mean concentrations of 9.6 ± 1.8, 492.4 ± 102.7 and 826.1 ± 189.4 µg/L at 0 h, and 7.4 ± 0.4, 337.5 ± 17.7, 680 ± 198.0 µg/L at each 24 h renewal period (representing 77, 69, and 82% of nominal concentrations at 24 h). The median measured concentrations over the whole exposure period were reported as 9, 415, and 753 µg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: stainless-steel tanks (10 liter)
- Aeration: No
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per vehicle control : 1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: well-aerated reconstituted tap water
- Culture medium different from test medium: Test medium same as culture medium.

OTHER TEST CONDITIONS
- Photoperiod: Light–dark 16:8 h

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Length (mm), weight (mg) and vitellogenin analysis (in whole body homogenates)

VITELLOGENIN ANALYSIS: Fish were individually homogenized in ice-cold assay buffer (Biosense, Bergen, Norway) in a 1:2 ratio wet weight:buffer volume, using a Ultra Turax homogenizer (IKA, HuberþCo.AG, Reinach, Switzerland). The homogenates were centrifuged at 10,000 3 g for 3 min at room temperature using a microcentrifuge The supernatant was assayed for vitellogenin (VTG) analysis using a quantitative heterologous carp enzyme-linked immunosorbent assay, which has been shown to be highly reliable for VTG determination in the fathead minnow.
Reference substance (positive control):
yes
Remarks:
100 ng/L 17beta estradiol

Results and discussion

Effect concentrationsopen allclose all
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
100 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
length
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
100 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
weight
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
415 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Vitellogenin induction
Remarks on result:
other: meas. is the median concentration
Duration:
14 d
Dose descriptor:
LOEC
Effect conc.:
415 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
length
Remarks on result:
other: meas. is the median concentration
Duration:
14 d
Dose descriptor:
LOEC
Effect conc.:
415 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
weight
Remarks on result:
other: meas. is median concentration
Details on results:
Toxic effects such as lethargy, uncoordinated swimming, loss of equilibrium, hyperventilation were observed at 753 µg/L 4-methylbenzylidene camphor (median measured concentration) and this test concentration was terminated after day 8 of exposure.
Results with reference substance (positive control):
There were no significant effects on length or weight with the positive control (E2) at 100 ng/L. There was significant induction of vitellogenin.
Reported statistics and error estimates:
After testing the data distribution for normality by using the Kolmogorov-Smirnov test, means of wet weight and total length of individual fish were calculated, and data were analyzed by analysis of variance (ANOVA) followed by a Dunnett’s Multiple Comparison test to compare the treatment means with respective controls. Means of VTG concentrations of individual fish were calculated, and data were analyzed with the non-parametric
Kruskal-Wallis test, followed by a Dunn’s Multiple Comparison test to compare the treatment means with respective controls. Statistical comparisons with the were made using the solvent control. The results are given as mean ± standard error of mean (SEM). Differences were considered significant at p <=0.05.

Applicant's summary and conclusion

Validity criteria fulfilled:
not specified
Conclusions:
4-methylbenzylidene camphor did not result in any oestrogenic effects in vivo in fish. Significant effects on length and weight at 415 ug/L 4-methylbenzylidene camphor (median measured concentration) were most likely due to systemic toxicity.
Executive summary:

The in vivo oestrogenic activity of 4-methylbenzylidene camphor was investigated in a 14-day vitellogenin (VTG) test with juvenile (sexually undifferentiated) fathead minnow (Pimephales promelas). 4-methylbenzylidene camphor was found not to exhibit any estrogenic activity in two yeast based estrogen receptor assays (human and rainbow trout) in the same study (see Sections 6.6 and 7.12). Ten fish per tank were exposed to concentrations of 10, 100, 500, 1000 4-methylbenzylidene camphor for 14 days in a static-renewal system. The test medium was renewed every 24 hours. The study included two controls and a solvent control (ethanol), as well as a positive control (100 ng/L E2). Samples of water were taken and the beginning of exposures (0 h) and just prior to water renewal (24 h) for analytical verification of the exposure concentrations. At the end of the exposure period fish were measured and weighed, and frozen prior to VTG analysis on whole body homogenates.

 

The nominal 10, 500 and 1000 µg/L exposures, had mean measured concentrations of 9.6 ± 1.8, 492.4 ± 102.7 and 826.1 ± 189.4 µg/L, respectively at 0 h, and 7.4 ± 0.4, 337.5 ± 17.7, 680 ± 198.0 µg/L, respectively after 24 h (representing 77, 69, and 82% of nominal concentrations at 24 h). The median measured concentrations over the whole exposure period were reported as 9, 415, and 753 µg/L. Toxic effects such as lethargy, uncoordinated swimming, loss of equilibrium, hyperventilation were observed at 753 µg/L 4-methylbenzylidene camphor (median measured concentration) and this test concentration was terminated after day 8 of exposure. Exposure to 4-methylbenzylidene camphor significantly reduced body length and weight of fish at median measured concentrations of 415 and 753 µg/L. 4-methylbenzylidene camphor exposure did not result in any significant VTG induction at any exposure concentration, which is consistent with the absence of in vitro activity found in yeast assays conducted by the same authors (see Section 6.6 and 7.12). The effects on length and weight are most likely due to systemic toxicity.

 

This study was assigned a reliability score of 2: reliable with restrictions for apical endpoints (length and body weight). The study was not conducted according to an international guideline, but was well-documented and scientifically acceptable. The VTG induction was assigned a reliability score of 3. The VTG induction was based on whole body homogenates of juvenile fish. The lack of specificity in terms of sex or tissue is expected to result in low sensitivity and makes the data difficult to interpret.