Hexanedioic acid, 1,6-bis(2-propylheptyl) ester

Endpoint summary

Additional information

In vitro studies

Reverse gene mutation assay (Ames)

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 Dipropylheptyladipat at concentrations of 33 - 5600 µg/plate in the precence 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) occured. Test substance precipitation was found from about 1000 µg/plate onward with and without metabolic activation. In conclusion, under the experimental conditions of this study, Dipropylheptyladipat was not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

Five reverse gene mutation assays (Zeiger et al. 1985; CMA/LBI20988; Kubo et al. 2002; Seed et al., 1982 and Simmon et al. 1977) in the bacteria strains of S.typhimurium (TA1535, TA1537, TA1538, TA98 and TA100) were determined with Diethylhexyladipat a structural analogue to Dipropylheptyladipat. In good agreement with the target chemical Dipropylheptyladipat, the source chemical Diethylhexyladipat revealed negative results in all five assays.

 

Micronucleus test in V79 cells (cytokinesis block method)

Dipropylheptyladipat 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 1 000 cells for each culture were analyzed for micronuclei, i.e. 2 000 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 test substance concentration. Therefore, concentrations at the border of test substance solubility in culture medium were investigated for cytogenetic damage. On the basis of the results of the present study, the test 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, Dipropylheptyladipat 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)

Diethylhexyladipat a structural analogue to Dipropylheptyladipat used for read-across assessement 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 occured 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 Diethylhexyladipat was not mutagenic in the system under the conditions employed.

 

This result is supported by a second mouse lymphoma cell forward mutation assay (CMA, 1982) conducted with the read across substance Diethylhexyladipat. Mouse lymphoma L5178Y cells were tested in concentrations up to 1000 nL/mL in the absence of metabolic activation and up to 200 nL/mL in the presence of metabolic activation. For the mutagenicity testing, cultures were exposed for 4 hours and growth for two or three days. At the end of the expression period cells for each selected dose were seeded in soft agar plates with selection medium and resistant (mutant) colonies were counted after 10 days incubation. The test item did not induce significant increases in the mutant frequency at the TK locus in L5178Y mouse lymphoma cells. Without metabolic activation, the test material was assayed up to the testing limit of 1000 nL/mL without inducing significant increases over the background (average of solvent and untreated control mutant frequencies). Moderately high toxicities were reached (percent relative growths, 60.2% to 21.4%). In the presence of metabolic activation, the test material was converted to a more toxic form, or forms, but no significant increases in the mutant frequencies were observed. Treatments from 15.6 nL/mL to 250 nL/mLwere assayed, and low to moderately high toxicities were induced (69.6% to 19.7% relative growths). Attempts to obtain higher toxicities were not successful due to a sharp toxicity curve. Diethylhexyladipat is therefore considered not mutagenic in the Mouse Lymphoma Forward Mutation Assay at concentrations that approach high toxicity.

 

Chromosome aberration assay

 

Chromosome aberrations were assessed in vitro in Chinese hamster ovary (CHO) cells with the source chemical for read-across i.e. Diethylhexyladipat (Galloway et al. 1987). The applied concentrations were in the range of 40-400 µg/mL with and without metabolic activation. No specific details were reported regarding exposure duration. Cells were collected by mitotic shake-off. Slides were stained with Giemsa and coded and 100 cells were scored from each of the three highest dose groups having sufficient metaphases for analysis. All types of aberrations were recorded separately, but for data analysis they were grouped into categories of simple (breaks and terminal deletions), complex (exchanges and rearrangements), other (includes pulverized chromosomes), and total. Gaps and endoreduplications were recorded but were not included in the totals.The aberration test with metabolic activation was negative, however there was some evidence for an increase without metabolic activation.

 

In a second assay with Diethylhexyladipat Chinese hamster ovary cells were determined for induction of sister chromatid exchange (SCE) with and without metabolic activation using concentrations of 40-400 µg/mL (Galloway et al. 1987). 5-Bromodeoxyuridine (BrdUrd) was added 2 hours after addition of the test item (without S9 mix) or immediately after the S9 mix plus chemical had been removed. The chemical treatment periods were approximately 25 hours without metabolic activation and 2 hours with metabolic activation. The total BrdUrd incubation time was 25-26 hours, with Colcemid present during the final 2-3 hours. Immediately before the cells were harvested, the cell monolayers were examined, and the degree of confluence and availability of mitotic cells were noted. Cells were collected, slides were prepared and evaluated. No further details about slide preparation and evaluation for Diethylhexyladipat were given. No SCE was observed in the absence of metabolic activation and equivocal results were shown for the experiment in the presence of metabolic activation.

 

The ambiguous results of the two studies were considered to be an incidental finding with no toxicological relevance. No evidence of a potential to induce chromosome aberrations in CHO cells was indicated for Diethylhexyladipat.

 

Transformation of Balb/3T3 cells

 

The morphological transforming potential of the source chemical Diethylhexyladipat was tested using mouse Balb/3T3 cells in culture (CMA/LBI20992). Thereby the ability to induce foci of transformed cells, recognized by dense, piled-up colonies on a monolayer of normal cells was evaluated in the presence and absence of metabolic activation systems. Cells were incubated with Diethylhexyladipat in concentrations up to 42 µL/mL. The test material did not induce the appearance of any transformed foci over the concentration range of 0.07 µg/mL to 42 µL/mL, corresponded to approximately 10.3% to 52% survival in the concomitant cytotoxicity test. Therefore, the test material is considered to be inactive in the Balb/3T3-rat liver cell mediated in vitro transformation assay.

 

In vivo studies

Mouse bone marrow micronucleus assay

Dipropylheptyladipat 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 test substance, dissolved in corn oil, was administered once orally to male animals at dose levels of 500 mg/kg, 1 000 mg/kg and 2 000 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 2 000 mg/kg body weight and in the vehicle controls. In the test groups of 1 000 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, 2 000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. The normocytes with and without micronuclei occurring per 2 000 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 (2 000 mg/kg body weight) at 48 hours sacrifice interval. Thus, bioavailabilty of the test substance in the target organ after oral administration was confirmed. According to the results of the present study, the single oral administration of XPB 115 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 Dipropylheptyladipat does not induce cytogenetic damage in bone marrow cells of NMRI mice in vivo.

The mouse bone marrow micronucleus assay (Shelby et al., 1993) was used to determine the genetic toxicity of Diethylhexyladipat. For the initial micronucleus (MN) test male B6C3F1 mice were injected intraperitoneal on three consecutive days with the test material in concentrations of 375, 750, 1500 and 2000 mg/kg bw. Mice were euthanized 24 hours after the third treatment. Bone marrow smears were prepared and slides were evaluated. Diethylhexyladipat was not judged to be positive in the micronucleus test and therefore was considered to be not genotoxic under condition of this study.

 

In a third mouse micronucleus test (CMA/LBI20996) Diethylhexyladipat was administered intraperitoneally to B6C3F1 mice (6 males and 6 females) in concentrations of 5000 mg/kg bw. Harvest of bone marrow was done 30 hours after injected for the acute treated group and 48 hours after the second injection for the multiple treated group. The two injections in the multiple treated group were 24 hours apart. Following extraction of the bone marrows, slides were prepared and evaluated. Diethylhexyladipat did not induce micronuclei in the bone marrow of B6C3F1 mice, and thus was not clastrogenic under the conditions of this assay.

 

Dominant lethal mutation assay

Dominant lethal mutations effects of Diethylhexyladipat were investigated in ICR mice (Singh et al., 1977). A single intraperitoneal dose of 0.5, 1.0, 5.0 or 10.0 mL/kg bw was given to males, which were then mated with 2 virgin females/week for 8 weeks. The number of early fetal deaths was determined for each female mouse. This parameter was used for as direct measure of dominant lethal mutations. The term postmeiotic was used synonymously with 1-3 week postinjection and premeiotic was used synonymously with 4-8 week postinjection. Dominant lethal mutations manifested by an increased number of early fetal deaths was found in higher dose levels in the premeiotic and postmeiotic stage of spermatogenesis in mice.

 

Conclusion

Dipropylheptyladipat did not induce genetic toxicity in the bacterial reverse gene mutation assay, the in vitro micronucleus test in V79 cells and an in vivo mouse bone marrow micronucleus test.

In addition, the read across substance Diethylhexyladipat, a structural analogue or surrogate to Dipropylheptyladipat (please refer to chapter 13 for details on the read-across approach) did also not induce genetic toxicity in several in vitro and in vivo studies. All reverse gene mutation assays in bacteria (Ames) and forward gene mutation assays in mammalian cells (mouse lymphoma) revealed negative results. Two chromosome aberration tests in CHO cells (aberration and sister chromatid exchange) showed ambiguous results, however the findings were considered to be incidental and no evidence of a potential to induce chromosome aberrations was indicated. Two micronucleus assays showed no micronuclei in the bone marrow of B6C3F1 mice and therefore, the test substance Diethylhexyladipat was considered to be not clastrogenic. In addition to the standard testing battery the in vitro transformation assay in Balb/3T3 cells and the in vivo dominant lethal mutation assay are avaible. Diethylhexyladipat did not induce transformed foci in Balb/3T3 cells under the test conditions. However,dominant lethal mutations manifested by an increased number of early fetal deaths were found in higher dose levels in the premeiotic and postmeiotic stage of spermatogenesis in mice. In the light of the consistently negative results of all other genotoxicity test and due to the fact that the dominant lethal mutation assay does not belong to the current standard testing battery single positive findings in this assay is considered to be without toxicological relevance.

Justification for selection of genetic toxicity endpoint
No study was selected because all in vitro and in vivo studies indicated that Dipropylheptyladipat is not genotoxic.

Short description of key information:
Reverse gene mutation assays in bacteria (Ames), forward gene mutation assays in mammalian cells (mouse lymphoma), chromosome aberration tests (aberration and sister chromatid exchange), in vitro micronucleus test, transformation assay in Balb/3T3 cells and in vivo micronucleus assays in mice indicated negative results for Dipropylheptyladipat and for the read across substance Diethylhexyladipat, a structural analogue to the target chemical Dipropylheptyladipat. The dominant lethal mutation test indicated a positive results for Diethylhexyladipat. However, in the light of all other negative results determined in the genotoxicity tests and the fact that the dominant lethal mutation test does not belong to the current standard testing battery, the single positive finding in this assay is considered to be without toxicological relevance.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available data, Dipropylheptyladipat does not have to be classified for genotoxicity according to Directive 67/548/EEC (DSD) and Regulation (EC) No 1272/2008 (CLP/GHS).