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AHTN has been assessed on its endocrine disrupting potential as part of the human and environmental hazard assessment. Based on a weight of evidence approach, as stated in the REACH Guidance, AHTN is not endocrine disrupting. This conclusion is affirmed in the EU risk assessment and confirmed by the SCHER (see IUCLID entry 13 "SCHER Environment, 2008").

Additional information

For AHTN, in vivo and in vitro assays indicate the following:

 

An in-vivo mouse uterotrophic assay demonstrated no uterotrophic activity at relatively high dietary levels (Seinen 1999).

An in-vitro study showed weak antagonistic behaviour in receptor binding assays using human embryonal kidney 293 (HEK293) cells transiently transfected with plasmids containing human and zebrafish estrogen receptor isoforms (Seinen 1999).

In an in-vivo study, transgenic zebrafish, containing a similar reporter gene construct as described in the above in vitro HEK293 experiment, were exposed to AHTN with and without estradiol. AHTN did not show any estrogenic effect. In contrast, a dose-dependent antagonistic effect on estradiol was observed. The authors concluded that no developmental disorders were or will be observed at the concentrations used in their transgenic zebrafish assay at actual concentrations (Schreurs 2004).

AHTN did not affect estrogen induced vitellogenin (Vtg) production (in-vitro), a sensitive marker of estrogenic activity, in cultured primary hepatocytes of carp (Cyprinus carpio; cited in Seinen 1999).

In and in-vitro study, the ecysteroid agonist and antagonist activity of AHTN was assessed in the Drosophila melanogaster BII-cell line (Breitholtz 2003). AHTN did not show specific agonistic or antagonistic activity in this bioassay up to the highest concentration tested (26 mg/L). The larval development rate of the N. spinipes was a result of pharmacological effects rather than endocrine-mediated toxicity. Acute and sub-chronic effects are close further supporting the claim that AHTN is not endocrine disrupting.

In an in-vitro competitive binding assay of AHTN with hepatic estrogen receptor(s) of rainbow trout, carp and the amphibian Xenopus leavis, AHTN did not demonstrate competitive inhibition of the binding of 17B-estradiol in the X. leavis assay. No competitive binding in the carp receptor-binding assay and only very weak binding of AHTN in the rainbow trout receptor binding assay was observed (Dietrich 2001).

 

The authors of the in vivo studies using transgenic zebrafish (Prof. Dr. W. Seinen and Dr. R.H.M.M. Schreurs) have indicated that the effects were observed at high concentrations and that the levels of AHTN of fish in the environment are much lower. Therefore, the authors indicate that AHTN is safe for use and for the environment, as biological effects that may be related to antiestrogenic activity are not anticipated.

 

The severity and magnitude of effects induced should also be taken into account. For example, potent estrogens halt the oestrous cycle and potent anti-androgens cause malformations of the male reproductive tract. Both are examples where fertility and reproduction are impaired. 

In the 90-day oral toxicity study on rats of sufficiently high doses and screening for reproductive parameters (enhanced OECD 408) no adverse effects on the reproductive parameters were observed (Lambert 1996).

A developmental toxicity test (OECD 414) was carried out and no toxicity was observed (Christian 1999). No adverse effects on embryo-foetal viability, growth or morphology occurred at the highest dosages of AHTN (50 mg/kg per day).

In a peri-postnatal rat study, pups were exposed in utero and the pups were followed for reproductive effects in the F1 and F2 generation (Ford 1997 and Jones 1996). No reproductive toxic or any other adverse effects were observed at the dams or their offspring at the highest dose tested (> 20 mg/kg bw).

The interaction of AHTN with multixenobiotic resistance (mxr) transporters was studied in gill tissue of the marine mussel Mytilus californianus. AHTN was shown to inhibit the efflux transporters in this tissue, which raised questions as the decreased transporter efflux renders the cell more accessible to other potential toxicants (Luckenbach 2005). However, as noted in the EU risk assessment, the mucous membrane plays a protective role, and removal of this membrane in this test system questions the relevance of these observations. Also the transporter activity in mussel gill is as sensitive as the effects observed in the standard toxicity tests with aquatic organisms. Thus, at the exposure level where the protective transporter efflux is decreased rendering the cell more accessible to other potential toxicants, other effects of the synthetic musks show up also in algae or fish tests studying development and growth. Also, in a subsequent letter to the editor, Salvito (2005) pointed out that measured environmental concentrations of the PCMs were 2-6 orders of magnitude lower than the effects concentration in this study and that the method was in development.

 

Conclusion of the weight of evidence approach: Based on a weight of evidence approach, as stated in the REACH Guidance, AHTN is not endocrine disrupting. This conclusion is affirmed in the EU risk assessment and confirmed by the SCHER (see IUCLID entry 13 "SCHER Environment, 2008").

The EU Risk Assessment referenced to:

[1] Seinen W, Lemmen JG, Pieters RHH, Verbruggen EMJ, Van der Burg B (1999). AHTN and HHCB show weak estrogenic – but no uterotrophic activity. Toxicology Letters 111, 161 – 168.

[2] Schreurs RHMM, Legler J, Artola-Garicano E, Sinnige TL, Lanser PH, Seinen W and Van der Burg B (2004). In vitro and in vivo antiestrogenic effects of polycyclic musks in zebrafish. Environ Sci & Technol. 38 (4), 997-1002.

[3] Breitholtz M, Wollenberger L, Dinan L (2003). Effects of four synthetic musks on the life cycle of the harpacticoid copepod Nitocra spinipes. Aquatic Toxicology. 63 (2), 103-118.

[4] Dietrich DR and Chou (2001). Y-J. Ecotoxicology of musks. In: American Chemical Society Symposium Series 791, Pharmaceutical and Personal Care Products in the Environment: Scientific and Regulatory Issues., ed.s C.G. Daughton and T. Jones-Lepp. American Chemical Society Washington DC.

[5] Lambert AH and Hopkins MN (1996). AHTN: 13–week oral (dietary) toxicity study in the rat with a 4 week treatment–free period. Toxicol Laboratories Limited, Ledbury, England. Toxicol report reference: RFA/5/95.

[6] Ford RA and Bottomley A (1997). A method for evaluation of the potential toxicity to the neonate from exposure to xenobiotics via mother's milk - application to three fragrance materials. The Toxicologist 36, No.1, Part 2, 367.

[7] Christian MS, Hoberman AM and Parker RM (1997). Oral (Gavage) developmental toxicity study of acetyl hexamethyl petroline (AHTN) in rats. Argus Research Laboratories, Horsham, PA. protocol nr. 1318-002.

[8] Jones K, Bottomley AM and Gopinath C (1996). AHTN: Effects on peri- and post natal development including maternal function in the rat. (Gavage administration) Report to the Research Institute for Fragrance Materials.

[9] Luckenbach T, Epel D (2004b). Nitromusks and polycyclic musk compounds as long-term inhibitors of cellulair xenobiotic defense systems mediated by multi-drug transporters. do1:10.1289/ehp.7301 (http://dx.doi.org/ online at 30 September 2004).