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Additional toxicological data

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Administrative data

Endpoint:
additional toxicological information
Remarks:
In vitro mechanistic data for endocrine activity
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:
No measurements of cytotoxicity were made, and, 4-MBC is toxic in the transgenic fish assay at the test concentrations where activity was observed. An appropriate reference standard was not used for anti-oestrogenic activity and therefore this data is assessed as unreliable
Justification for type of information:
Experimental information for endocrine activity discussion (report in Section 13).

Data source

Reference
Reference Type:
publication
Title:
Estrogenic activity of UV filters determined by an in vitro reporter gene assay and an in vivo transgenic zebrafish assay
Author:
Schreurs R, Lanser P, Seinen W, van der Burg B
Year:
2002
Bibliographic source:
Arch. Toxicol., Regulatory Toxicology 76:257-261

Materials and methods

Type of study / information:
Investigation of the in vitro oestrogenic and anti-oestrogenic activity of 4-methylbenzylidene camphor, in a HEK293 reporter gene assay.
Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
In vitro oestrogenic and anti-oestrogenic activity of 4-methylbenzylidene camphor, in a HEK293 reporter gene assay.
- Short description of test conditions: The cell line was cultured at 37oC and in an atmosphere of 7.5% CO2
-Cell line: Human embryonal kidney 293 (HEK293) cells were obtained from the American Type Culture Collection (ATCC, Rockville, Md., USA). Cells were maintained in a 1:1 mixture of Dulbecco’s modified Eagle’s medium (DMEM) and Ham’s F12 medium (DF;Life Technologies Inc., Gaithersburg, Md., USA) supplemented with 7.5% fetal calf serum (FCS; Integro, Linz, Austria).
-hERalpha and hERbeta gene expression: The cell line contained stable hERa and hERb transfectants of HEK293 cells.
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 (Darmstadt, Germany).

Results and discussion

Any other information on results incl. tables

4-methylbenzylidene camphor was found to activate the hERalpha receptor at 10 µM and 100 µM, which resulted in luciferase activity of approximately 65% and 55%, respectively. of maximal E2 induction. 4-MBC was also found to activate the hERβ receptor at 10 µM and 100 µM, which resulted in luciferase activity of approximately 10% and 75%, respectively of maximal E2 induction. The test concentrations where activity was observed are above the water solubility limit for 4-MBC.

There was significant repression of hERβ activity based on the percentage of E2 induction at 0.1, 1.0 and 10 µM 4-methylbenzylidene camphor with luciferase activity of approximatly 60, 50 and 60%, respectively, however, there was no significant effect at 100 µM which had activity of approximatly 70%. The results did not show a clear dose response. There was no effect on repression of hERalpha activity. The data for anti-oestrogenic activity is not reliable because it was not reported based on a reference standard substance.

Applicant's summary and conclusion

Conclusions:
The study provides some evidence for oestrogenic activity at the hERalpha and beta receptors. The test concentrations where effects were observed are above the water solubility limits for the substance of 1.3 mg/L. 4-methylbenzylidene camphor is also toxic in a transgenic fish assay reported in the same study at the test concentrations where oestrogenic activity was observed.
Executive summary:

The in vitro oestrogenic and anti-oestrogenic activity of 4-methylbenzylidene camphor was investigated in a HEK293 reporter gene assay. Human embryonic kidney 293 (HEK293) cells containing stably transfected hERalpha and hERβ were used. Binding of a (xeno-)oestrogen to the estrogen receptors (ERs) results in ER-transactivation and luciferase gene induction. Luciferase activity was measured in cell lysates using a scintillation counter. This study also evaluated the in vivo oestrogenic activity in a transgenic fish assay of 4-methylbenzylidene camphor reported in Section 6.6.1. The test concentrations (given in figures of the results section) were 0.1, 1, 10 and 100 µM (0.025, 0.25, 2.5 and 25 mg/L). Each test concentration was run in triplicate and the results were the mean of at least two experiments. The authors did not report whether cytotoxicity was controlled for in the study design and toxicity was observed in a transgenic fish assay at test concentrations where activity was observed in this assay. 4 -methylbenzyllidene camphor was found to activate the hERalpha receptor at 10 µM and 100 µM, which resulted in luciferase activity of approximately 65% and 55%, respectively, of maximal E2 induction. 4-methylbenzylidene camphor was also found to activate the hERβ receptor at 10 µM and 100 µM, which resulted in luciferase activity of approximately 10% and 75%, respectively, of maximal E2 induction. The data for anti-oestrogenic activity is not reliable because it was not reported based on a reference standard substance, i.e. the study did not include a positive control for anti-oestrogenicity.

This study was assigned a reliability score of 2: reliable with restrictions for determination of oestrogenic activity in vitro. This experiment was not conducted according to an international guideline, but used a well-established approach. An appropriate reference standard was not used for anti-oestrogenic activity and cytotoxicity was not evaluated, therefore the results from the anti-oestrogenic assays are not reliable.