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

Endpoint summary

Additional information

Key studies:

HHCB (>99% pure) in acetone was tested in the Ames test in absence or presence of Aroclor-induced rat liver S9 at a dose ranging from 10 to 5000 mg/plate according to OECD guideline 471 using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, TA1538 and Escherichia Coli strain WP2 UVRA and appropriate positive controls. Based on preliminary range-finding studies, doses of 10 (not tested in confirmation assay), 33, 100, 333, 1000, 3333 (only tested in confirmation assay) or 5000 µg HHCB/plate were used. Slight precipitation was seen at the three highest doses (≥333 µg/plate). All dose levels of HHCB, acetone (negative control) and positive controls were plated in triplicate. All positive controls gave positive responses to the systems within acceptable ranges. No significant increase in the number of revertant colonies was observed for HHCB at any dose with any of the six strains with or without activation. (San et al., 1994, Api and San, 1999a)

A cytogenetic assay with Chinese Hamster ovary cells (CHO-K1) was conducted on HHCB (purity >99%) according to OECD Guideline 473. Concentrations of 5, 10 and 20 μg HHCB/ml were used without metabolic activation, using 4/20, 20/20 and 44/44 hr exposure/harvest periods. In the study with metabolic activation (S9 from rat liver induced by Aroclor 1254), dose levels of 8.7, 17.3 and 34.5 μg HHCB/ml were tested for the 4-hr period with a 20-hr harvest time and dose levels of 22.6, 28.2 and 30.0 μg/ml for the 4-hr period with a 44-hr harvest time. At the 20 and 44-hr harvest times, the cells were assessed for structural chromosome aberrations, and at the 44-hr harvest time, also for numerical chromosome aberrations. The mitotic index was significantly lowered at the highest dose in all cases. N-methyl-N’-nitro-N-nitrosoguanidine was used as a positive control in the non-activated study and benzo(a)pyrene in the activated study. Positive controls caused increases in structural (significantly) aberrations in all cases. No significant increase in structural or numerical chromosome aberrations was observed with or without activation with HHCB at any dose. HHCB was concluded to be negative for chromosome aberrations in this test. (Curry and Putman, 1995, Api and San, 1999a)

An in vitro unscheduled DNA synthesis (UDS) assay in accordance with OECD guideline 482 was conducted with HHCB (purity >99%) in acetone in primary rat hepatocytes at concentrations of 0.15, 0.50, 1.5, 5.0, 15 mg/ml (50-5000 µg/ml proved too toxic to test). The positive control 7,12-dimethylbenz(a)anthracene induced a significant increase in the average net nuclear grain count over controls. No increase in net nuclear grain count was seen for HHCB up to and including 15 µg/ml although this dose did induce significant cytotoxicity as determined by LDH leakage. 50 µg/ml proved too toxic to be evaluated. (San and Sly, 1995, Api and San, 1999a)

 

Additional information:

A second test is available to assess the genotoxic potential of HHCB in bacteria (Mersch-Sundermann et al., 1998a), as well as a SOS chromotest in Escherichia coli (Mersch-Sundermann et al., 1998b). Both studies showed no genotoxic potential of HHCB. Furthermore, two in vitro micronucleus tests are available (both Kevekordes et al., 1997), as well as an in vivo micronucleus test (Api and San, 1999a, Gudi and Ritter, 1997). All three studies showed no significant increase of micronuclei after HHCB-treatment. Additionally, a sister-chromatid exchange (SCE) is available (Kevekordes et al., 1998). No effects were observed after treatment with HHCB.

An overview of the available mutagenicity studies is presented in the table below.

 

Type	Species	Activation	Doses	Results	GLP	OECD	Reference
In vitro Bacterial Reverse Mutation Assay	Salmonella typhimurium (TA98, 100, 1535, 1537, 1538) & E. coli (WP2 UVRA)	w & w/o S9	10, 33, 100, 333, 1000, 3333, 5000 µg/plate	negative	Yes	471	Api and San, 1999, San and Sly, 1994
In vitro Bacterial Reverse Mutation Assay	Salmonella typhimurium (TA97, 98, 100 and 102)	w & w/o S9	5, 16.6, 50, 166.6, or 500 µg/plate	negative	No		Mersch-Sunderman, 1998a
In vitro SOS induction	E. coli PQ37	w & w/o S9	0.39, 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50µg/assay	negative	No		Mersch-Sunderman, 1998b
In vitro cytogenetic assay	Chinese hamster ovary cells	w & w/o S9	9, 17, 34µg/ml & 23, 28, 30 µg/ml	negative	Yes	473	Api and San, 1999a, Curry and Putman, 1995
In vitro micronucleus test	Human lymphocytes	w & w/o S9	0.05, 0.49, 4.85, 48.5, 97, 194µM (=0.013, 0.13, 1.25, 12.5, 25, 50 µg/ml)	negative	No		Kevekordes et al., 1997
In vitro micronucleus test	Human hepatoma cells	None added, some inherent	0.1, 0.97, 9.7, 97, 194, 387 µM (=0.036, 0.25, 2.5, 25, 50, 100 µg/ml)	negative	No		Kevekordes et al., 1997
In vitro sister chromatid exchange test	Human lymphocytes	w & w/o S9	0.025, 0.25, 2.43, 24.25, 48.5, 97µM (= 0.007, 0.07, 0.6, 6, 12.5, 25 µg/ml)	negative	No		Kevekordes et al., 1998
In vitro unscheduled DNA synthesis	Primary rat hepatocytes	Inherent	0.15, 0.5, 1.5, 5, 15, 50µg/ml	negative	Yes	482	Api and San, 1999, San and Sly, 1994
In vivo micronucleus test	mice		380, 750, 1500 mg/kg bw	negative	yes	474	Api and San, 1999, Gudi and Ritter, 1997

 

 

Conclusion and discussion:

HHCB has been tested in a wide array of in vitro tests and in an in vivo mouse micronucleus test. In vitro, HHCB was negative in gene mutation tests with bacteria, in an SOS chromotest with bacteria, in SCE and micronucleus tests with human cells, in an UDS test with primary rat hepatocytes and in a chromosome aberration assay in CHO cells. HHCB also did not induce significant chromosome aberrations in the in vivo micronucleus test. Hence, it can be judged that HHCB is a non-genotoxic substance.

 

(Summary in line with EU-RAR, 2008)

Justification for selection of genetic toxicity endpoint
Three required in vitro genotoxicity tests were performed in accordance with OECD and under GLP conditions.

Short description of key information:
In vitro:
- gene mutation in bacteria (Ames test): negative with and without metabolic activation (OECD471)
- cytogenicity (chromosome aberration test): negative with and without metabolic activation (OECD 473)
- gene mutation in mammalian cells (UDS test): negative with an without metabolic activation (OECD482)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available in vitro and in vivo mutagenicity studies, HHCB does not have to be classified as mutagenic in accordance with 67/548/EEC (DSD) and 1272/2008/EC (CLP).