Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Additional information

Justification for grouping of substances and read-across

The PFAE Linear (Polyfunctional Aliphatic Ester) category consists of 16 substances, well-defined mono-constituent substances as well as related UVCB substances, respectively with varying fatty alcohol chain lengths and branching. The distinguishing feature of this category of chemicals is that they are diester derivatives of common dicarboxylic acids: namely adipic (C6), azelaic (C9) and sebacic (C10) acids. The alcohol portion of the diesters generally falls in the C3-C20 carbon number range, including linear and branched, even and odd numbered alcohols. 

In order to avoid the need to test every substance for every endpoint, the category concept is applied for the assessment of environmental fate and environmental and human health hazards. Thus where applicable, environmental and human health effects are predicted from adequate and reliable data for source substance(s) within the group by interpolation to the target substances in the group (read-across approach) applying the group concept in accordance with Annex XI, Item 1.5, of Regulation (EC) No 1907/2006. In particular, 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 grouping of chemicals and read-across is provided in the technical dossier (see IUCLID Sections 7.1 and 13) and within Chapter 5.1 of the CSR.Endpoint specific data matrix:

Table 1: Genetic toxicity.

ID#

CAS

Genetic Toxicity in vitro: Gene mutation in bacteria

Genetic Toxicity in vitro: cytogenicity in mammalian cells

Genetic Toxicity in vitro: gene mutation in mammalian cells

Genetic toxicity in vivo

1

6938-94-9 (a)

RA: CAS 105-99-7

RA: CAS 105-99-7

Negative

RA: CAS 105-99-7

2

105-99-7

Negative

Positive + S9

RA: CAS 103-23-1
RA: CAS 33703-08-1
RA: CAS 6938-94-9

Negative

3

110-33-8

Negative

RA: CAS 105-99-7
RA: CAS 103-23-1

RA: CAS 103-23-1

Negative
RA: CAS 105-99-7
RA: CAS 103-23-1

4

1330-86-5

RA: CAS 105-99-7
RA: CAS 33703-08-1

RA: CAS 105-99-7

RA: CAS 103-23-1
RA: CAS 33703-08-1
RA: CAS 6938-94-9

RA: CAS 105-99-7
RA: CAS 16958-92-2

5

123-79-5 (b)

--

--

--

--

6

103-23-1

Negative

Negative
Positive –S9

Negative

Negative

7

68515-75-3

Negative (c)

--

--

--

8

33703-08-1

Negative

--

Negative

--

9

16958-92-2

Negative

RA: CAS 103-23-1

RA: CAS 103-23-1
RA: CAS 33703-08-1
RA: CAS 6938-94-9

Negative

10

85117-94-8

--

--

--

--

11

103-24-2

Negative

Negative

RA: CAS 103-23-1

--

12

897626-46-9

RA: CAS 103-24-2
RA: CAS 69275-01-0

RA: CAS 103-24-2

Negative

RA: CAS 69275-01-0

13

7491-02-3

RA: CAS 109-43-3
RA: CAS 122-62-3

RA: CAS 105-99-7
RA: CAS 103-24-2

RA: CAS 103-23-1
RA: CAS 33703-08-1
RA: CAS 6938-94-9
RA: CAS 897626-46-9

RA: CAS 105-99-7

14

109-43-3

Negative
RA: CAS 122-62-3

RA: CAS 105-99-7
RA: CAS 103-24-2

RA: CAS 103-23-1
RA: CAS 33703-08-1
RA: CAS 6938-94-9
RA: CAS 897626-46-9

RA: CAS 105-99-7

15

122-62-3

Negative
RA: CAS 103-24-2

RA: CAS 103-24-2

RA: CAS 103-23-1
RA: CAS 33703-08-1
RA: CAS 6938-94-9
RA: CAS 897626-46-9

--

16

69275-01-0

Negative

--

--

Negative

(a) Category members subject to registration are indicated in bold font. Only for these substances a full set of experimental results and/or read-across is given.

(b) Substances not subject to registration are indicated in normal font. Lack of data for a given endpoint is indicated by “--“.

(c) Original data source is not available and thus not used for read-across.

 

Genetic toxicity (mutagenicity) in bacteria in vitro

Diester of Adipic acid

CAS 105-99-7

The potential mutagenicity of Dibutyl adipate (CAS 105-99-7) was investigated in two bacterial reverse mutation assays (Ames test).

One bacterial gene mutation assay (Ames test) with the test substance was conducted in compliance with OECD guideline 471 and under GLP conditions (Shibuya, 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.

In a further study the potential mutagenicity of Dibutyl adipate was investigated in a bacterial reverse mutation assay (Ames test) similar to OECD guideline 471 and under conditions of GLP (Schröder, 1996). In two independent experiments, the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 were exposed to the test substance at concentrations ranging from 8 to 5000 µg/plate (first experiment) and 50 to 1000 µg/plate (second experiment), respectively. Both experiments were performed in the absence and presence of a liver microsomal activation system (S9 mix) using the plate incorporation assay. Cytotoxicity occurred at concentrations ≥ 250 µg/plate in the absence of S9 mix. No increase in the mean number of revertants was observed in any tester strain at any concentration tested. 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, the test substance was considered to be non-mutagenic in the selected strains of S. typhimurium (TA 98, TA 100, TA 1535, TA 1537 and TA 1538) in the presence and absence of metabolic activation.

In summary, Dibutyl adipate, tested up to the limit concentration of 5000 µg/plate was non-mutagenic in S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 and E. coli WP2 uvrA in the presence and absence of metabolic activation.

 

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 (Hafner, 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 103-23-1

The potential mutagenicity of Bis(2-ethylhexyl) adipate (CAS 103-23-1) was tested in a bacterial gene mutation assay similar to OECD 471 (Zeiger et al. 1985). In two independent experiments, the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 were exposed to the test substance at concentrations of 100, 333, 1000, 3333 and 10000 µg/plate. Both experiments were performed in the absence or presence of a liver microsomal activation system (S9 mix) using the preincubation method (20 min preincubation time). No cytotoxicity was noted in the in treated bacteria strains when compared to controls. Precipitation of the test substance was observed at 10000 µg/plate in all test strains in the repeat experiment. The mean number of revertant colonies per plate was not increased at any test concentration. The positive control substances for each tester strain showed the expected results.

Under the conditions of this assay, Bis(2-ethylhexyl) adipate was found to be non-mutagenic in S. typhimurium strains (TA 1535, TA 1537, TA 98 and TA 100), both in the presence and absence of S9 mix.

 

CAS 33703-08-1

In a GLP-conform study similar to OECD guideline 471, the potential mutagenicity of Diisononyl adipate (CAS 33703-08-1) in bacteria was investigated in the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 (McKee et al., 1986). In two independent experiments, bacteria were treated with concentrations of 10, 50, 100, 500 and 1000 µg/plate, either in the presence or absence of hamster or rat liver S9 mix. Exposure to the test substance at the indicated concentrations did not increase the mean number of revertants in bacteria There was no indication of cytotoxicity based on the number of spontaneous revertants up to and including 1000 µg/plate. The positive controls included in the assay demonstrated the efficiency of the assay.

Under the conditions of this bacterial gene mutation assay, Diisononyl adipate was found not to be mutagenic in the selected strains of S. typhimurium (TA 98, TA 100, TA 1535, TA 1537 and TA 1538) in the presence and absence of metabolic activation.

 

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 (Thompso, 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.

 

Diester of Azelaic acid

CAS 103-24-2

The potential mutagenicity of Bis(2-ethylhexyl) azelate (CAS 103-24-2) was tested in a bacterial gene mutation assay (Ames test) according to OECD 471 and in compliance with GLP (Miwa, 2004). Based on the results of a preliminary cytotoxicity experiment, the Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 and Escherichia coli WP2 uvrA were treated with the test substance at concentrations of 312.5, 625, 1250, 2500 and 5000 µg/plate using the plate incorporation assay. The test was performed in two independent experiments, both with and without metabolic activation system (S9-mix). No cytotoxicity was observed in the two experiments up to the limit concentration of 5000 µg/plate. Colourless oil drop-like and oil membrane-like precipitations were observed on the surface of agar at all test concentrations. The mean number of revertant colonies was not increased at any test concentration compared to control. The positive controls included showed the expected results.

Under the conditions of this experiment, Bis(2-ethylhexyl) azelate did not induce mutations in the bacterial gene mutation assay in the selected strains of S. typhimurium (TA 98, TA 100, TA 1535 and TA 1537) and in E. coli WP2 uvrA in the presence and absence of metabolic activation.

 

Diester of Sebacic acid

CAS 109-43-3

A bacterial gene mutation assay (Ames test) with Dibutyl sebacate (CAS 109-43-3) was performed similar to OECD guideline 471 (Hachiya and Takizawa, 1994). In this experiment, the direct plate incorporation procedure was conducted with the Salmonella typhimurium strains TA 97, TA 98, TA 100 and TA 102 and Escherichia coli WP2 uvrA pKM 101 at concentrations of 200, 500, 1000, 2000, 5000 and 10000 µg/plate. All bacteria strains were exposed for a period of 44-46 h, both in the presence and absence of metabolic activation (S9 mix). No cytotoxicity and no increase in the mean number of revertants per plate were observed at any test concentration. No positive controls were included to confirm the efficiency of the assay. 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 97, TA 98, TA 100 and TA 102) and in E. coli WP2 uvrA pKM 101.

In a further Ames test, with only limited detail available (reliability 4), the potential mutagenicity of Dibutyl sebacate was investigated similar to OCED guideline 471, the Salmonella typhimurium strains S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100 were treated with 5 concentrations up to 3600 µg/plate using the plate incorporation assay in the presence or absence of liver microsomal activation system (Wild et al., 1983). The mean number of revertants per plate was not increased at this test concentration compared to controls. The positive controls included showed the expected results. Based on these results, the test substance was not considered to be mutagenic in the presence and absence of metabolic activation in the selected strains of S. typhimurium (TA 1535, TA 1537, TA 1538, TA 98 and TA 100) under the conditions of this bacterial mutation assay.

In summary, Dibutyl sebacate was non mutagenic in the S. typhimurium strains TA 97, TA 98, TA 100, TA 102, TA 1535, TA 1537 and TA 1538 and E. coli WP2 uvrA pKM 101 in the presence and absence of metabolic activation.

 

CAS 122-62-3

The potential mutagenicity of Bis(2-ethylhexyl) sebacate (CAS 122-62-3) was tested in a bacterial gene mutation assay similar to OECD 471 (Zeiger et al. 1985). In two independent experiments, the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 were exposed to the test substance at concentrations of 100, 333, 1000, 3333 and 10000 µg/plate. Both experiments were performed in the absence or presence of a liver microsomal activation system (S9 mix) using the preincubation method (20 min preincubation time). No cytotoxicity was noted in the in treated bacteria strains when compared to controls. Precipitation of the test substance was observed at 3333 and 10000 µg/plate in S. typhimurium strain TA1535 only. The mean number of revertant colonies per plate was not increased at any test concentration. The positive control substances for each tester strain showed the expected results.

Under the conditions of this assay, Bis(2-ethylhexyl) sebacate was found to be non-mutagenic in the selected S. typhimurium strains (TA 1535, TA 1537, TA 98 and TA 100), both in the presence and absence of S9 mix.

 

CAS 69275-01-0

A bacterial gene mutation assay (Ames test) with Bis(2-octyldodecyl) sebacate (CAS 69275-01-0) was performed similar to OECD guideline 471 and in compliance with GLP (Wallat, 1986). Two independent experiments were performed, both in the presence or absence of metabolic activation (S9 mix), in the Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 with test substance concentrations of 8, 40, 200, 1000 and 5000 µg/plate using the plate incorporation assay. Cytotoxicity was only observed in the second experiment in S. typhimurium strain TA1537 at the limit concentration of 5000 µg/plate in the absence of S9 mix. No increase in mean revertant numbers was observed in any bacterial strain after exposure to the test substance at any test concentration in both experiments. The positive controls showed the expected results and thus confirmed the efficiency of the assay.

Under the conditions of this assay, Bis(2-octyldodecyl) sebacate did not induce mutagenicity in the selected strains of S. typhimurium (TA 1535, TA 1537, TA 1538, TA 98 and TA 100) in the absence and presence of metabolic activation .

 

Genetic toxicity (cytogenicity) in mammalian cells in vitro

Diester of Adipic acid

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 (Tanaka, 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 (Honarvar, 2002), where no clastogenicity in male and female NMRI mice was detected (for details see Genetic toxicity in vivo).

 

CAS 103-23-1

Two studies are available investigating the clastogenic potential of Bis(2-ethylhexyl) adipate (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 (Howard, 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 and100 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 controls 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.

 

In the second study an in vitro mammalian chromosome aberration test was performed with Bis(2-ethylhexyl) adipate in Chinese hamster ovary (CHO-W-B1) cells similar to OECD guideline 473 (Galloway, 1987). Based on a preliminary toxicity test, concentrations of 40, 130 and 400 μg/mL were used to analyse chromosomal aberrations after exposure to the test substance for 2 h (harvest time: 14 h) in the presence and for 14 h in the absence of metabolic activation (S9 mix), respectively. A statistically significant increase in the number of cells with chromosomal aberrations compared to solvent control was observed after 14 h exposure to the test substance at a concentration of 400 µg/plate in the absence of metabolic activation. In contrast, exposure to the test substance for 2 h in the presence of metabolic activation did not significantly increase the number of aberrant cells compared to the solvent control. No data on cytotoxicity was presented. The positive controls showed the expected increase in the rate of chromosome aberrations, thus indicating the sensitivity of the assay.

In conclusion, Bis(2-ethylhexyl) adipate was clastogenic in Chinese hamster ovary (CHO-W-B1) in the absence of metabolic activation. The results of this in vitro study could not be reproduced in vivo in a Mouse Bone Marrow Micronucleus Test performed with Bis(2-ethylhexyl) adipate in male B6C3F1 mice similar to OECD guideline 474 (Shelby, 1993), where no clastogenicity in male mice was detected (for details see Genetic toxicity in vivo).

 

Diester of Azelaic acid

CAS 103-24-2

The clastogenic potential of Bis(2-ethylhexyl) azelate (CAS 103-24-2) was assessed in an in vitro mammalian chromosome aberration test in Chinese hamster lung (CHL/IU) cells according to OECD guideline 473 and under GLP conditions (Miwa, 2004). In a preliminary cytotoxicity test, 50% growth inhibition was observed after 6 h short-term treatment with the test substance at concentrations of 1859.0 µg/mL with S9 mix and at 1555.5 µg/mL without S9 mix. After continuous treatment for 24 h, cell growth was inhibited by 50% at 534.2 µg/mL without S9 mix. Based on these results, concentrations ranging from 150 to 2400 µg/mL (with and without S9 mix) and 37.5 to 600 µg/mL (without S9 mix) were selected for chromosome analysis after 6 and 24 h treatment in the main study, respectively. No increase in the number of cells with chromosomal aberrations was observed compared to controls in any of the experiments performed. The test substance was cytotoxic at ≥ 600 µg/mL after 6 h short-term treatment and at ≥ 300 µg/mL after 24 h continuous treatment. Visible precipitation of the test substance was observed at concentrations ≥ 1200 µg/mL, but did not interfere with chromosomal analysis. The positive controls included during short-term and continuous exposure showed the expected results and thus verified the sensitivity of the assay.

Under the conditions of this experiment, Bis(2-ethylhexyl) azelate was considered not to be clastogenic in Chinese hamster lung (CHL/IU) cells in the presence or absence of metabolic activation.

 

Genetic toxicity (mutagenicity) in mammalian cells in vitro

Diester of Adipic acid

CAS 6938-94-9

An in vitro mammalian cell gene mutation assay with Diisopropyl adipate was performed according to OECD guideline 476 (Morris, 2013). Mutations at the HPRT locus of Chinese hamster Ovary (CHO) cells in the presence of metabolic activation (S9 mix) were investigated in two independent experiments at test substance concentrations ranging from 71.88 to 2300 µg/mL (4 h exposure). In two further experiments, cells were exposed to the test substance at concentrations ranging from 35 to 560 µg/mL(4 h expoisure) or 8.98 to 575 µg/mL (24 h exposure), respectively, in the absence of metabolic activation. Following treatment, cells were cultured for 7 days to allow growth and expression of induced mutants, followed by a selection period of 14 days in the presence of 6-Thioguanine (6-TG) selective medium. The test substance did not induce a significant increase in the mutant frequency at any test substance concentration in the presence or absence of S9 mix. Precipitation of the test item was observed at 210 µg/mL (4 h exposure) and at 431.25 µg/mL (24 h exposure) without metabolic activation and at 575 µg/mL with metabolic activation, respectively. Cytotoxicity was noted only in the absence S9 mix at 560 µg/mL (4 h exposure) and at 431.25 µg/mL and above (24 h exposure). The positive controls significantly increased mutant frequency, demonstrating the sensitivity of the test system and the efficacy of the metabolic activation system. In conclusion, the test substance was not mutagenic to Chinese hamster Ovary (CHO) cells in the presence or absence of metabolic activation under the conditions of this test.

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, 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 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.

 

CAS 33703-08-1

An in vitro mammalian cell gene mutation assay was performed with Diisononyl adipate (CAS 33703-08-1) according to OECD guideline 476 and under conditions of GLP (McKee et al., 1986). Mutations at the TK locus of mouse-lymphoma L5178Y cells were investigated at test substance concentrations ranging from 7.5-100 µL/mL in the absence of metabolic activation (S9 mix) or 5.6-100 µL/mL in the presence of metabolic activation. Assuming a test substance density of 0.92 g/mL, these concentrations approximately corresponded to mass concentrations of 6.8-92 mg/mL and 5.1-92 mg/mL, respectively. After exposure to the test substance, cytotoxicity was observed at concentrations ≥ 10 µL/mL with relative total growth values ≤ 37%. After a 2-day expression period of the cultures, the resistance to 5-trifluorothymidine (TFT) was determined in all experiments. The test substance did not induce a significant increase in the mutant frequency at any dose concentration. The positive controls significantly increased mutant frequency, demonstrating the sensitivity of the test system and the efficacy of the metabolic activation system.

In conclusion, Diisononyl adipate did not induce the mutant frequency in mouse-lymphoma L5178Y cells, neither in the presence nor in the absence of a metabolic activation system, under these experimental conditions.

 

Diester of Azelaic acid

CAS 897626-46-9

An in vitro mammalian cell gene mutation assay was performed with Bis(2-octyldodecyl) azelate (CAS 897626-49-9) according to OECD guideline 476 and under conditions of GLP (Wollny, 2013). Mutations at the HPRT locus of Chinese hamster lung fibroblasts (V79) were investigated at test substance concentrations ranging from 81.3 - 2600 µg/mL in the absence and presence of metabolic activation (S9 mix). Two independent experiments were performed with 2 parallel cultures for each condition, respectively. In the first experiment, exposure time was 4 h with and without S9 mix and in the second experiment exposure time was 4 h with S9 mix and 24 h without S9 mix. A solvent control (THF) and positive controls were included. After exposure to the test substance, cytotoxicity was determined by evaluating the cloning efficiency directly after exposure (survival) and after the expression time (viability). After the 7-day expression period of the cultures, the resistance to 6-thioguanine (6-TG) was determined in all experiments. Phase separation was observed from 350 µg/mL. To avoid analysis of too many concentrations showing phase separation, the highest dose (2600 µg/mL) was not continued. An increase in mutant frequency was seen in the second experiment without metabolic activation after 24 h treatment at 650 and 1300 µg/mL. As this effect only occurred in one of the two cultures, showed no concentration-relation and was moreover accompanied by cytotoxic effects (cell viability < 50%), this finding was not considered biologically relevant. The positive controls significantly increased mutant frequency, demonstrating the sensitivity of the test system and the efficacy of the metabolic activation system.

In conclusion, Bis(2-octyldodecyl) azelate, did not induce the mutant frequency in Chinese hamster lung fibroblasts (V79), neither in the presence nor in the absence of a metabolic activation system, under these experimental conditions.

 

Genetic toxicity in vivo

Diester of Adipic acid

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 (Honarvar, 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 110-33-8

The genetic toxicity of Dihexyl adipate (CAS 110-33-8) in vivo was investigated in a Mammalian Erythrocyte Micronucleus Test similar to OECD guideline 474 (Farrow, 1984). In this study, male and female Sprague Dawley rats were once treated with the test substance in corn oil at dose levels of 1000, 3000 and 10000 mg/kg bw via oral gavage, and bone marrow was sampled either 6, 12, 24 and 48 h after treatment. Six animals per sex, group and sampling time were used for treatment with the test substance or the vehicle, respectively. No increase in micronuclei frequency was observed in bone marrow cells of males and females up to the maximum dose of 10000 mg/kg bw after post-exposure periods of 6, 12 and 24 h. Since no evidence was seen that the compound caused mitotic delay, the slides from the 48 h sacrifice were not analysed for chromosome aberrations.

Based on the results of this micronucleus test, Dihexyl adipate was considered to be non-clastogenic to male and female Sprague Dawley rats under the conditions of this experiment.

 

CAS 103-23-1

The in vivo clastogenicity of Bis(2-ethylhexyl)adipate (CAS 103-23-1) was investigated in a Mouse Bone Marrow Micronucleus Test similar to OECD guideline 474 (Shelby, 1993). Male B6C3F1 mice (5 per group) were intraperitoneally treated with the test substance diluted in corn oil at dose levels of 375, 750, 1500 and 2000 mg/kg bw/day for 3 consecutive days. Five further animals received the vehicle only and served as control group. 24 hours after the last treatment, bone marrow smears were prepared and the number of micronucleated polychromatic cells (PCE) among 2000 PCE was assessed. Furthermore, the percentage of PCE among 200 erythrocytes was determined to assess cytotoxicity. No increases in the frequency of micronuclei in polychromatic erythrocytes compared to the vehicle control were observed up to a dose level of 2000 mg/kg bw/day and the percentage of PCE compared to vehicle control did not indicate a dose-related increase in cytotoxicity in the bone marrow.

Based on the results of this study, Bis(2-ethylhexyl) adipate did not induce clastogenicity in male B6C3F1 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 (Skinner, 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.

 

Diester of Sebacic acid

CAS 69275-01-0

An in vivo Mammalian Erythrocyte Micronucleus Test according to OECD Guideline 474 was performed with Bis(2-octyldodecyl) sebacate (CAS 69275-01-1) in CFW 1 mice under conditions of GLP (Wallat, 1986). Based on a preliminary range-finding toxicity test, 7 animals per sex and group received the test substance in oleum arachidis DAB8 once via oral gavage at dose levels of 1000 and 5000 mg/kg bw. Furthermore, a high dose group, consisting of 3 subgroups with each 7 animals per sex, was gavaged with the test substance at 10000 mg/kg bw. A control group of 7 animals per sex was treated with the vehicle only. Cyclophosphamide was used as positive control substance and administered intraperitoneally at a dose level of 10 mg/kg bw to 7 animals per sex. Femoral bone smears of animals assigned to the vehicle control, positive control, 1000 and 5000 mg/kg bw dose groups were prepared 24 h after exposure to the test substance, whereas preparation of femoral bone smears of the three subgroups receiving 10000 mg/kg bw was performed 24, 48 and 72 h post-exposure, respectively. After preparation, the number of micronuclei per 1000 polychromatic erythrocytes was counted. In addition, the ratio of polychromatic erythrocytes to normochromatic cells was determined to assess cytotoxicity. Clinical signs of toxicity of slightly reduced activity and ruffled fur occurred in males and females at dose levels of 5000 and 10000 mg/kg bw only within the first 20 h after exposure. Since no increases in the frequency of micronuclei of polychromatic cells were observed in the femoral bone marrow of CFW 1 mice exposed to test substance at 10000 mg/kg bw at any preparation time point, scoring of micronuclei was not performed at the lower dose levels. The ratios of poly- to normochromatic cells did not indicate cytotoxicity in bone barrow of animals treated at 10000 mg/kg bw. The positive control substance significantly increased the number of normochromatic and polychromatic erythrocytes with micronuclei in males and females.

Based on the results of this micronucleus test in erythrocytes, Bis(2-octyldodecyl) sebacate was considered to be non-clastogenic to male and female CFW 1 mice under the conditions of this experiment.

 

Conclusion for genetic toxicity

In summary, 10 studies investigating the genetic mutation in bacteria in vitro are available within the PFAE linear category for Dibutyl adipate (CAS 105-99-7), Dihexyl adipate (CAS 110-33-8), Bis(2-ethylhexyl) adipate (CAS 103-23-1), Diisononyl adipate (CAS 33703-08-1), Bis(tridecyl) adipate (CAS 16958-92-2), Bis(2-ethylhexyl) azelate (CAS 103-24-2), Dibutyl sebacate (CAS 109-43-3), Bis(2-ethylhexyl) sebacate (CAS 122-62-3)and Bis(2-octyldodecyl) sebacate (CAS 69275-01-0), all providing negative results. Furthermore, no cytogenicity in mammalian cells in vitro was observed for the category member Bis(2-ethylhexyl) azelate (CAS 103-24-2). In contrast, Dibutyl adipate (CAS 105-99-7) and Bis(2-ethylhexyl) adipate (CAS 103-23-1) showed a clastogenic potential in the presence of S9 mix in Chinese hamster lung cells or in the absence of S9 mix in Chinese hamster ovary cells, respectively, which was not confirmed in subsequent Micronucleus Tests in vivo. Two mouse lymphoma assays for the category members Bis(2-ethylhexyl) adipate (CAS 103-23-1) and Diisononyl adipate (CAS 33703-08-1) and two HPRT assays for the category members Bis(2-octyldodecyl) azelate (CAS 897626-46-9) and Diisopropyl adipate (CAS 6938-94-9) showed negative results indicating no mutagenicity in mammalian cells in vitro. No genetic toxicity in vivo was observed in Micronucleus Assays for Bis(2-ethylhexyl) adipate (CAS 103-23-1),Dibutyl adipate (CAS 105-99-7), Dihexyl adipate (CAS 110-33-8), Bis(tridecyl) adipate(CAS 16958-92-2) and Bis(2-octyldodecyl) sebacate CAS 69275-01-0).

Therefore, no properties for genetic toxicity were observed within the PFAE linear group for any member.

 

References

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Lilja, J. et al. (2005). Esterification of propanoic acid with ethanol, 1-propanol and butanol over a heterogeneous fiber catalyst. Chemical Engineering Journal, 115(1-2): 1-12.

Liu, Y. et al. (2006). A comparison of the esterification of acetic acid with methanol using heterogeneous versus homogeneous acid catalysis. Journal of Catalysis 242: 278-286.

Radzi, S.M. et al.(2005). High performance enzymatic synthesis of oleyl oleate using immobilised lipase from Candida antartica. Electronic Journal of Biotechnology 8: 292-298.

Takahashi et al. (1981). Elimination, distribution and metabolism of di- (2-ethylhexyl) adipate (DEHA) in rats. Toxicology, 22, 223-233.

Zhao, Z. (2000). Synthesis of butyl propionate using novel aluminophosphate molecular sieve as catalyst. Journal of Molecular Catalysis 154(1-2): 131-135.


Short description of key information:
No properties for genetic toxicity were observed within the PFAE linear group for any member.

Endpoint Conclusion: No adverse effect observed (negative)

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 group concept is applied to the members of the PFAE linear Category, data will be generated from representative reference substance(s) within the category to avoid unnecessary animal testing. Additionally, once the group concept is applied, substances will be classified and labelled on this basis.

Therefore, based on the group concept, all available data on genetic toxicity do not meet the classification criteria according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.

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