Registration Dossier

Administrative data

Key value for chemical safety assessment

Additional information

Due to a lack of experiments with the test substance and the existence of a structural analog (read-across substance) part of or the complete data was derived from the read-across substance. For details on the read-across reliability please refer to IUCLID Section 13. 

In vitro studies

Reverse gene mutation assay (Ames)

The study tested the mutagenicity of the registered substance. The study was conducted according to OECD TG 471. The Ames test was performed as a standard plate test and as preincubation test with and without the addition of a metabolizing system obtained from rat liver (S9 mix) using the Salmonella strains TA 1535, TA 100, TA 1537, TA 98 and Escherichia coli WP2 uvrA. The test substance was applied in concentrations of 0, 33, 100, 333, 1000, 2500 and 5000 µg/plate. The experiments were done in triplicate. No bacteriotoxic effects were observed. Precipitation was detected from 1000 µg/plate onward with and without metabolic activation. All tests and strains showed a negative effect for mutagenicity. Therefore the test substance was considered non mutagenic under the corresponding conditions.

In a reverse gene mutation assay the bacteria strains TA 1535, TA 1537, TA 98 and TA 100 of S. typhimurium and of Escherichia coli WP2 uvrA were exposed to the read-across substance Bis(2-propylheptyl) hexanedioate at concentrations of 33 - 5600 µg/plate in the presence and the absence of metabolic activation applying both the standard plate and the pre-incubation method (BASF SE 2013). A biologically relevant increase in the number of his+ or trp+ revertants was not observed in both independent experiments either without S9 mix or after the addition of a metabolizing system. Indeed a slight decrease in the number of his+ and trp + revertants from about 1000 µg/plate onward (standard plate test) and from about 2800 µg/plate onward (preincubation test) occurred. Read-across substance precipitation was found from about 1000 µg/plate onward with and without metabolic activation. In conclusion, under the experimental conditions of this study, the read-across substance was not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

 

Micronucleus test in V79 cells (cytokinesis block method)

The read-across substance Bis(2-propylheptyl) hexanedioate was assessed (BASF SE 2014) for its potential to induce micronuclei in V79 cells in vitro (clastogenic or aneugenic activity). Two independent experiments were carried out, with and without the addition of liver S9 mix from induced rats (exogenous metabolic activation) with test concentrations of 0 - 200 µg/mL. A sample of at least 1000 cells for each culture were analyzed for micronuclei, i.e. 2000 cells for each test group. The vehicle controls gave frequencies of micronucleated cells within our historical negative control data range for V79 cells. Both positive control substances, EMS and cyclophosphamide, led to the expected increase in the number of cells containing micronuclei. In this study, no cytotoxicity indicated by reduced cell count or proliferation index (CBPI) was observed up to the highest applied read-across substance concentration. Therefore, concentrations at the border of read-across substance solubility in culture medium were investigated for cytogenetic damage. On the basis of the results of the present study, the read-across substance did not cause any biologically relevant increase in the number of cells containing micronuclei either without S9 mix or after adding a metabolizing system. Thus, the read-across substance is considered not to have a chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.

 

Forward gene mutation (mouse lymphoma assay)

A structural analogue to the registered substance, Bis(2-ethylhexyl) hexanedioate, was tested in concentrations up to 5000 µg/mL for its mutagenic potential in the mouse lymphoma cell forward mutation assay (Mc Gregor et al., 1988). Mouse lymphoma L5178Y cells were exposed to the test item for 4 hours, then cultured for 2 days before plating in soft agar with or without trofluorothymidine (TFT) in the presence and absence of metabolic activation systems. Precipitation of the test item was observed at 1000 µg/mL, but testing was continued up to 5000 µg/mL. In the absence of S9 mix there was no indication of a mutagenic response in two experiments. In the presence of S9 mix, one experiment similarly failed to show any mutagenic effect, while significant increases in mutant fraction occurred in a second experiment. The LOED in this experiment was 2000 µg/mL, higher than the precipitation concentration. Significant toxicity was observed in all experiments. It was concluded that the structural analogue to the primary read-across substance was not mutagenic in the system under the conditions employed.

   

In vivo studies

Mouse bone marrow micronucleus assay

The read-across substance Bis(2-propylheptyl) hexanedioate was assessed for its potential to induce chromosomal damage (clastogenicity) or spindle poison effects (aneugenic activity) in NMRI mice using the micronucleus test method (BASF SE 2014). For this purpose, the read-across substance, dissolved in corn oil, was administered once orally to male animals at dose levels of 500 mg/kg, 1000 mg/kg and 2000 mg/kg body weight in a volume of 10 mL/kg body weight in each case. The animals were sacrificed and the bone marrow of the two femora was prepared 24 and 48 hours after administration in the highest dose group of 2000 mg/kg body weight and in the vehicle controls. In the test groups of 1000 mg/kg and 500 mg/kg body weight and in the positive control group, the 24-hour sacrifice interval was investigated only. After staining of the preparations, 2000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. The normocytes with and without micronuclei occurring per 2000 polychromatic erythrocytes were also recorded. As vehicle control, male mice were administered merely the vehicle, corn oil, by the same route and in the same volume as the animals of the dose groups, which gave frequencies of micronucleated polychromatic erythrocytes within the historical vehicle control data range. The positive control substance, cyclophosphamide, led to the expected increase in the rate of polychromatic erythrocytes containing only small micronuclei. A clear inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected at the highest recommended test substance dose (2000 mg/kg body weight) at 48 hours sacrifice interval. Thus, bioavailability of the read-across substance in the target organ after oral administration was confirmed. According to the results of the present study, the single oral administration of the read-across substance did not lead to any relevant increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was within the range of the concurrent vehicle control in all dose groups and at all sacrifice intervals and within the range of the historical vehicle control data. Thus, under the experimental conditions of this study the read-across substance does not induce cytogenetic damage in bone marrow cells of NMRI mice in vivo.

 

Conclusion

The registered substance and the read-across substances did not induce genetic toxicity in the bacterial reverse gene mutation assay. The read-across substance Bis(2-propylheptyl) hexanedioate did not induce genetic toxicity in the in vitro micronucleus test in V79 cells and an in vivo mouse bone marrow micronucleus test.

In addition, the other read-across substance Bis(2-ethylhexyl) hexanedioate (please refer to chapter 13 for details on the read-across approach) did also not induce genetic toxicity.


Justification for selection of genetic toxicity endpoint
4 studies (all negative) were used to evaluate the genotoxic potential of the test substance.

Short description of key information:
The registered substance and the read-across substance Bis(2-propylheptyl) hexanedioate did not induce genetic toxicity in the bacterial reverse gene mutation assay. The read-across substance Bis(2-propylheptyl) hexanedioate did not induce genetic toxicity in the in vitro micronucleus test in V79 cells and an in vivo mouse bone marrow micronucleus test.
In addition, another structural analogue to the registered substance, Bis(2-ethylhexyl) hexanedioate, did also not induce genetic toxicity.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008 as amended for the seventh time in Regulation (EC) No 2015/1221. As a result based on an Ames test and the read-across data the substance is not considered to be classified for genetic toxicity.