Bis(1-methylheptyl) adipate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro gene mutation study in bacteria: negative (read-across from CAS 110-33-8)

In vitro cytogenicity / chromosome aberration study in mammalian cells: positive (read-across from CAS 105-99-7) / negative (read-across from 103-23-1)

In vitro gene mutation study in mammalian cells: negative (read-across from CAS 103-23-1)

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus: negative (read-across from CAS 105-99-7 and CAS 16958-92-2)

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Justification for read across

Data on cytogenicity in mammalian cells, on gene mutation in bacterial and mammalian cells and in vivo genetic toxicity data with bis(1-methylheptyl) adipate (CAS 108-63-4) were not available. The assessment was therefore based on studies conducted with analogue substances as part of a read across approach, which is in accordance with Regulation (EC) No. 1907/2006, Annex XI, 1.5. For each specific endpoint the source substance(s) structurally closest to the target substance is/are chosen for read-across, with due regard to the requirements of adequacy and reliability of the available data. Structural similarities and similarities in properties and/or activities of the source and target substance are the basis of read-across. A detailed justification for the analogue read-across approach is provided in the technical dossier (see IUCLID Section 13).

Genetic toxicity (mutagenicity) in bacteria in vitro

CAS 110-33-8

The potential mutagenicity of dihexyl adipate (CAS 110-33-8) was investigated in a bacterial reverse mutation assay (Ames test) according to OECD guideline 471 and under conditions of GLP (Hydrotox, 2012). Five strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98, TA 100 and TA 102) were tested in the presence and absence of a liver metabolic activation system (S9-mix) at concentrations of 16, 50, 158, 500, 1581 and 5000 µg/plate in the first experiment and at concentrations of 480, 860, 1540, 2780 and 5000 µg/plate in the second experiment. In both experiments, cells were exposed to the test substance for a period of 48 h using the plate incorporation assay. No cytotoxicity occurred and no increase in the mean number of revertants was observed in any tester strain up to 5000 µg/plate compared to controls. The positive controls included in the assay showed the expected results and verified the efficiency of the assay. Based on the results of this experiment, dihexyl adipate was considered to be non-mutagenic in the selected strains of S. typhimurium (TA 1535, TA 1537, TA 98, TA 100 and TA 102) in the presence and absence of metabolic activation.

CAS 105-99-7

The potential mutagenicity of dibutyl adipate (CAS 105-99-7) was investigated in a bacterial gene mutation assay (Ames test) conducted in compliance with OECD guideline 471 and under GLP conditions (MHLW, 1996). The assay was performed with the Salmonella strains TA 98, TA 100, TA 1535 and TA 1537 and the Escherichia coli strain WP2 uvrA at test substance concentrations of 312.5, 625, 1250, 2500 and 5000 µg/plate both in the absence and in the presence of liver microsomal activation system (S9 mix) using the plate incorporation assay. In two independent experiments, cytotoxicity was evident as reduction in bacterial background growth in TA 100 only at a concentration of 5000 µg/plate. The mean number of revertant colonies was not increased at any concentration tested and the positive and negative controls included showed the expected results in each experiment. Under the experimental conditions reported, the test substance did not induce mutations in the bacterial mutation assay in the absence and presence of metabolic activation in the selected strains of S. typhimurium (TA 98, TA 100, TA 1535 and TA 1537) and in E. coli WP2 uvrA.

CAS 16958-92-2

In a GLP-conform study performed according to OECD guideline 471, the mutagenicity of bis(tridecyl) adipate in bacteria was investigated (Chemtura, 2013). The S. typhimurium strains 535, TA 1537, TA 98, TA 100 and E. coli WP2 were tested in two independent experiments at concentrations ranging from 50 to 5000 µg/plate both in the presence or absence of metabolic activation (S9 mix). The first experiment was performed using the plate incorporation procedure, whereas the second experiment was conducted according to the preincubation method (20 min preincubation time). No cytotoxicity was observed in any of the tester strains up 5000 µg/plate, but precipitation of the test substance was observed at concentration ≥ 1500 µg/plate both in the presence of absence of the metabolic activation system, but did not interfere with the scoring of revertant colonies. Exposure to the test substance in the presence or absence of metabolic activation did not increase the mean number of revertants in any strain of bacteria tested compared to the solvent controls in both experiments. The positive and negative (solvent) controls included in the assay yielded the expected results. Under the conditions of this bacterial mutation assay, the test substance was found to be non-mutagenic in the selected strains of S. typhimurium and E.coli in the presence and absence of metabolic activation.

Genetic toxicity (cytogenicity) in mammalian cells in vitro

CAS 105-99-7

An in vitro mammalian chromosome aberration test was performed with dibutyl adipate (CAS 105-99-7) in Chinese hamster lung (CHL/IU) cells according to OECD guideline 473 and in compliance with GLP (MHLW, 1996). Based on a preliminary toxicity test, concentrations of 700, 1300 and 2600 μg/mL were used to analyse chromosomal aberrations after exposure to the test substance for 6 h in the presence and for 24 and 48 h in the absence of metabolic activation (S9 mix), respectively. In addition, cells were exposed to 12, 23 and 46 µg/mL of the test substance for 6 h in the absence of S9 mix. A statistically significant increase in the number of cells with chromosomal aberrations compared to solvent control was observed after 6 h exposure to the test substance at concentrations of 700 and 1300 µg/plate in the presence of metabolic activation. Under this test conditions, treatment with 2600 µg/mL also increased the number of aberrant cells compared to the control, but this effect was not statistically significant due to the increased cytotoxicity at this concentration. In contrast, exposure to the test substance for 6, 24 and 48 h in the absence of metabolic activation did not significantly increase the number of aberrant cells compared to the solvent control. The positive controls showed the expected increase in the rate of chromosome aberrations, thus indicating the sensitivity of the assay.

In conclusion, dibutyl adipate was clastogenic in Chinese hamster lung (CHL/IU) cells in the presence of metabolic activation. The results of this in vitro study could not be reproduced in a subsequent in vivo Mammalian Erythrocyte Micronucleus Test performed with dibutyl adipate in NMRI mice according to OECD Guideline 474 (RCC, 2002), where no clastogenicity in male and female NMRI mice was detected (for details see Genetic toxicity in vivo).

CAS 103-23-1

An in vitro mammalian chromosome aberration test was performed with bis(2-ethylhexyl) adipate in a primary human lymphocyte cell culture from two donors similar to OECD Guideline 473 (ICI, 1989). The occurrence of chromosome aberrations was investigated after treatment of the cells for 3 h in the presence and absence of metabolic activation (rat liver S9-mix) at test substance concentrations of 0, 10, 50 and 100 µg/mL diluted in DMSO. The highest dose was the maximum achievable as determined by the solubility of the test sample in DMSO. Cells were harvested 28 h after start of exposure and 100 cells in metaphase per blood culture and concentration were evaluated for the incidence of breaks, fragments and minutes, multiple damages, interchanges and others. A reduction in the mitotic index compared to solvent control to 35% (without S9-mix) and 18% (with S9-mix) in donor 1 at 100 µg/mL and to <= 50% (without S9-mix) in donor 2 at 10 and 50 µg/mL indicated cytotoxicity of the test substance. The positive control substances cyclophosphamide and mitomycin C significantly increased the rate of chromosome aberrations indicating the sensitivity of the assay. No statistically or biologically significant increases in chromosomal damage were seen at any of the dose levels tested, in either donor, in the presence or absence of S9-mix. Under the conditions of this assay, bis(2-ethylhexyl) adipate was not clastogenic to cultured human lymphocytes in vitro.

Genetic toxicity (mutagenicity) in mammalian cells in vitro

CAS 103-23-1

An in vitro mammalian cell gene mutation assay with bis(2-ethylhexyl) adipate (CAS 103-23-1) was performed similar to OECD guideline 476 (McGregor et al., 1988). Mutations at the TK locus of mouse-lymphoma L5178Y cells in the presence of metabolic activation (S9 mix) were investigated in two independent experiments at test substance concentrations ranging from 312.5 to 5000 µg/mL. In two further experiments, cells were exposed to the test substance at concentrations ranging from 1000 to 5000 µg/mL in the absence of metabolic activation. The treatment of cells in all experiments included an exposure period of 4 h, followed by an expression period of 48 h and a selection period of 11-14 days in the presence of 5-trifluorothymidine (TFT). The test substance did not induce a significant increase in the mutant frequency at any test substance concentration in the absence of S9 mix. No significant increase in mutant frequency was observed in the first experiment in the presence of S9 mix. In contrast, a statistically significant increase in the mutant frequency was observed in cells treated at concentrations ≥ 2000 µg/mL in the second experiment with S9 mix. However, since no dose-response relationship was observed and precipitation was evident at concentrations of 1000 µg/mL, the slight significant increase in mutant frequencies in the presence of S9 mix was not considered to be of biological relevance. In addition, significant cytotoxicity, as indicated by a reduction in relative total growth, was noted in all experiments at concentrations ≥ 1000 µg/mL, both with and without S9 mix. The positive controls significantly increased the mutant frequency, demonstrating the sensitivity of the test system and the efficacy of the metabolic activation system. In conclusion, the test substance did not induce the mutant frequency in mouse-lymphoma L5178Y cells in the presence and absence of metabolic activation.

 

Genetic toxicity in vivo

CAS 105-99-7

An in vivo Mammalian Erythrocyte Micronucleus Test was performed with dibutyl adipate (CAS 105-99-7) in NMRI mice according to OECD Guideline 474 and under conditions of GLP (RCC, 2002). In a preliminary acute toxicity study, 2 animals per sex were exposed to the test substance at a single maximum dose of 2000 mg/kg bw in olive oil via gavage. No signs of acute toxicity were observed at intervals of 1, 2-4, 6, 24, 30 and 48 h after administration, except for a reduction in spontaneous activity in all 4 animals 1 h post-treatment. Since these effects were reversible within 24 h, a maximum dose of 2000 mg/kg bw was chosen in the main experiment. In this study, groups of 6 animals per sex received the test substance in olive oil once via oral gavage at dose levels of 500, 1000 and 2000 mg/kg bw. A similar constituted control group received the vehicle only. Treated and control animals were sacrificed 24 h after application of the test substance for preparation of bone marrow smears. An additional group receiving the test substance at 2000 mg/kg bw was sacrificed after 48 h. At least 2000 polychromatic erythrocytes of the femoral bone marrow were scored in 5 animals per sex and group to determine the frequency of micro-nucleated erythrocytes. No increases in the frequency of micronuclei in polychromatic erythrocytes compared to the vehicle control were observed up to a dose of 2000 mg/kg bw, both at the 24 and the 48 h sampling interval. The ratio between polychromatic and normochromatic erythrocytes did not indicate treatment-related cytotoxicity in the bone marrow of the animals. At 2000 mg/kg bw, clinical signs of toxicity occurred and included a reduction of spontaneous activity in 12/12, abdominal position in 3/12, eyelid closure in 4/12 and ruffled fur in 8/12 animals 1 h after test substance application. However, these effects were fully reversible within 24 h post-treatment. The positive control substance (40 mg/kg bw cyclophosphamide) significantly increased the number of polychromatic erythrocytes with micronuclei after 24 h exposure, and thus confirmed the sensitivity of the assay. Under the conditions of this experiment, dibutyl adipate did not induce clastogenicity in male and female NMRI mice in the micronucleus assay.

CAS 16958-92-2

The clastogenic potential of bis(tridecyl) adipate (CAS 16958-92-2) was investigated in an in vivo Mammalian Erythrocyte Micronucleus Test in Sprague-Dawley rats similar to OECD Guideline 474 (Mobil, 1985). The test substance was applied to the clipped dorsal skin of 10 animals per sex and group at dose levels of 800 and 2000 mg/kg bw/day. Treatment of the animals was performed daily for 5 days/week over a period of 13 weeks. A similar constituted control group remained untreated and served as controls. At the end of exposure, the femoral bone marrow and peripheral blood smears of 5 animals per sex and dose were prepared and the incidence of micronucleated cells per 1000 polychromatic or normochromatic erythrocytes were scored in the respective tissues. In addition, the number of polychromatic and normochromatic erythrocytes was determined and a ratio of both was calculated in order to determine cytotoxic effects of the test substance. The obtained ratios of poly- to normochromatic cells did not indicate cytotoxicity in bone barrow and peripheral blood of treated animals. The frequency of micronuclei in polychromatic and normochromatic erythrocytes of the femoral bone marrow and peripheral blood of treated animals was not increased at any dose level compared to controls after 13-week dermal exposure. Under the conditions of this experiment, bis(tridecyl) adipate did not induce clastogenicity in male and female Sprague Dawley rats in the micronucleus assay.

Overall conclusion for genetic toxicity

The results of the available in vitro mutagenicity studies in bacterial and mammalian cells were consistently negative. However, dibutyl adipate (CAS 105-99-7) was clastogenic in Chinese hamster lung (CHL/IU) cells in the presence of metabolic activation, whereas bis(2-ethylhexyl) adipate (CAS 103-23-1) did not show clastogenic properties to cultured human lymphocytes in vitro. Therefore, in vivo data were taken into account for the assessment of the genotoxic potential of the target substance. The positive results of the in vitro study with dibutyl adipate (CAS 105-99-7) could not be reproduced in a subsequent in vivo Mammalian Erythrocyte Micronucleus Test performed with the same test material in NMRI mice according to OECD Guideline 474, where no clastogenicity in male and female NMRI mice was detected. Moreover, a further Mammalian Erythrocyte Micronucleus Test in Sprague-Dawley rats with bis(tridecyl) adipate (CAS 16958-92-2) did not induce clastogenicity in male and female Sprague Dawley rats. Overall, no genotoxic potential is expected for the target substance bis(1-methylheptyl) adipate (CAS 108-63-4).

Justification for classification or non-classification

According to Article 13 of Regulation (EC) No. 1907/2006 "General Requirements for Generation of Information on Intrinsic Properties of substances", information on intrinsic properties of substances may be generated by means other than tests e.g. from information from structurally related substances (grouping or read-across), provided that conditions set out in Annex XI are met. Annex XI, "General rules for adaptation of this standard testing regime set out in Annexes VII to X” states that “substances whose physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a group, or ‘category’ of substances. This avoids the need to test every substance for every endpoint". Since the analogue concept is applied to bis(1-methylheptyl) adipate (CAS 108-63-4), data will be generated from data for reference source substance(s) to avoid unnecessary animal testing. Additionally, once the analogue read-across concept is applied, substances will be classified and labelled on this basis.

Therefore, based on the analogue read-across approach, the available data on genetic toxicity do not meet the classification criteria according to Regulation (EC) No. 1272/2008 and are therefore conclusive but not sufficient for classification.