1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylindeno[5,6-c]pyran

Specific investigations: other studies

Description of key information

HHCB is assessed for its potential endocrine activities by focusing on estrogen-, androgen-, thyroid and steroid activation (EATS)-related mode of actions (MOA), using IUCLID information and the information presented in the Risk Management Option Analysis (RMOA, 2019).

Human health:

Available In vitro data on HHCB related to  estrogenicity, androgenicity, thyroidogenicity and steroidogenicity were found in literature. There are some anti-estrogen, anti-androgen and anti-progesterone effects seen in vitro. However, the specificity of these observed effects in relationship to endocrine activities seem to be questionable because they occur generally at relatively high doses (1-10 uM).

Several in vivo repeated dose and reproductive toxicity assays are available: 90-day study (OECD TG 408, 1997); Dose range finding (DRF) for the Extended One Generation Reproductive Toxicity test (EOGRT, 2021), the Extended one generation reproduction (EOGRT) study and the developmental toxicity studies in rat and rabbit (OECD TG 414, 1997 and 2020, respectively). For systemic effects liver and thyroid are the target organ showing increases in relative weights and hypertrophy in both organs. An NOAEL for these effects is recommended to be at 92 mg/kg bw (EOGRT, 2021). Effects on the liver suggest liver enzyme induction by HHCB, which increases the clearance of thyroid hormone T4. A well-known feedback mechanism to maintain T4 level in circulation may have resulted in thyroid hypertrophy and weight increase (Curran and DeGroot, 1991). HHCB did not affect T4 and TSH levels in blood or the pituitary gland, which supports this proposed mode of action.

No fertility and no developmental or teratogenic effects were found in the standard toxicity tests available. There are decreases in body weight seen in male pups up to 12% in the DRF for the EOGRT and in the EOGRT. This effect was not considered adverse because 1) no fetal weight decreases were seen in the developmental toxicity studies in rat and rabbit ((OECD TG 414); 2) the body weight effect was seen in the F1 cohorts but not in F2 generations and were still within the historical control values (EOGRT, OECD TG 443) and 3) there were no other health, viability or reproductive effects. Accordingly, there were no suggestive endocrine-related effects identified in the in vivo studies of HHCB that could be connected to a human health concern. This means that the active findings (mostly at high concentrations) in in vitro tests are not reflected in the in vivo apical endpoints.

 

Environment:

In vitro results from Zebrafish receptors transferred in human cell-lines show some anti-estrogenicity and anti-steroidogenicity generally at high concentrations.

In vivo results: In Zebrafish these anti-estrogenic effects are seen at high concentrations (1 uM=258 ug/l) when also general toxic effects occur based on the NOECs of all fish: 68 ug/l (0.26 uM). In the Medaka study estrogenic effects such as increase in Vitellogenin are seen at concentrations just below 96h-LC50 (950 ug/l), that is at 500 ug/l. Also In these two fish species there is no clear pattern on either anti-estrogenic or estrogenic effects.

For invertebrates, standard and non-standard tests are available of which none have specifically investigated endocrine activity. Apical endpoint such as reproduction may have an endocrine background, but this is speculative. To have some indication on whether reproductive toxicity derives from general toxicity or has another basis, the following was assessed.

In studies where reproduction is affected at the same concentrations as growth or feeding rate, this indicates general toxic effects. Where reproduction is affected at lower concentrations, a mechanism of action is not clear. In the standard toxicity tests where reproduction and development are measured, but no other general toxic parameters scored, no indication of mechanism can be given either, like the long term Daphnia study. In the non-standard sediment tests with the New Zealand mud snail and Capitella, presented in the RMOA (2019), generally toxicity effects are present at the same or lower concentration than reproductive effects, e.g. feed reduction in the New Zealand mud snail. Or the finding on decreased number of eggs in Capitella sp. is not confirmed by effects on brood size or population effects. These two non-standard toxicity studies lack some methodological details too for accurate assessment.

Based on fish and invertebrate studies, there are currently no indications for endocrine-related effects for environmental species as shown in these long-term studies, including those where juveniles are mated and reproduction is measured. Therefore, additional studies are not needed. Though, some mostly anti-estrogenic effects in vitro are seen at high concentrations in fish (1-10 uM; 258-2580 ug/l). These concentrations also cause acute and long-term effects in fish or other aquatic organisms. Therefore, general toxicity effects cover for potentially (anti)estrogenic effect.

In addition, absence of relevant endocrine activity findings in mammals can support the absence of endocrine activity in aquatic vertebrates (and invertebrates) because there is a high level of conservation of the endocrine system across the taxonomic groups (ECHA guidance, 2018). No endocrine effects were observed in the EOGRT study in rats, further supporting the absence of endocrine activity in the environment.

 

Conclusion

Overall, for human health and environment there are some in vitro indications for mainly anti-estrogen/androgen/ steroid endocrine effects in both mammals and fish, which are not seen in vivo or only in the presence of other toxicity related effects. Therefore, there is no causal relation established between the in vitro endocrine effects and the in vivo apical endpoints, on which basis there is no concern for endocrine activity.

Additional information

A full ED report is present in the attachment.