Registration Dossier

Administrative data

Key value for chemical safety assessment

Additional information

Justification for grouping of substances and read-across

There are no data available for the genetic toxicity of Fatty acids, C16-18 and C18-unsatd., hexaesters with dipentaerythritol (CAS# 68604-38-6). In order to fulfil the standard information requirements set out in Annex VIII, 8.4, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006, read-across from structurally related substances was conducted.

In accordance with Article 13 (1) of Regulation (EC) No 1907/2006, "information on intrinsic properties of substances may be generated by means other than tests, provided that the conditions set out in Annex XI are met.” In particular for human toxicity, information shall be generated whenever possible by means other than vertebrate animal tests, which includes the use of information from structurally related substances (grouping or read-across).

Having regard to the general rules for grouping of substances and read-across approach laid down in Annex XI, Item 1.5, of Regulation (EC) No 1907/2006 whereby substances may be predicted as similar provided that their physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity.

 

Overview for genetic toxicity

 

In vitro

In vivo

CAS

Bacterial gene mutation

Cytogenicity in mammalian cells in vitro

Mammalian gene mutation

CAS 68604-38-6 (a)

Target substance

RA: CAS 85116-93-4

RA: CAS 85186-89-6

RA: CAS 189200-42-8

RA: CAS 647028-25-9

RA: CAS 403507-18-6

RA: CAS 189200-42-8

RA: CAS 647028-25-9

RA: CAS 85186-89-6

RA: CAS 68424-31-7

RA: CAS 189200-42-8

CAS 68424-31-7 (b)

--

--

--

negative

CAS 85116-93-4

negative

--

--

--

CAS 85186-89-6

negative

--

negative

--

CAS 403507-18-6

--

negative

--

--

CAS 189200-42-8

negative

negative

--

negative

CAS 647028-25-9

negative

negative

--

--

 (a) Substances subject to the REACh  Phase-in registration deadline of 31 May 2013 are indicated in bold font. Only for this substance a full set of experimental results and/or read-across is given.

 (b) Substances that are either already registered under REACh or not subject to the REACh  Phase-in registration deadline of 31 May 2013 are indicated in normal font. Lack of data for a given endpoint is indicated by “--“.

 

The above mentioned substances are considered to be similar on the basis of structural similarity resulting in similar properties and/or activities. The available endpoint information is used to predict the same endpoints for Fatty acids, C16-18 and C18-unsatd., hexaesters with dipentaerythritol (CAS# 68604-38-6)

A detailed analogue approach justification is provided in the technical dossier (see IUCLID Section 13).

Discussion

There are no studies available assessing the genetic toxicity of the substance to be registered. To evaluate potential genetic toxicity in vitro and in vivo data from Fatty acids, C16-18, esters with pentaerythritol (CAS# 85116-93-4), Fatty acids, C8-18 and C18-unsatd., esters with trimethylolpropane (CAS# 85186-89-6), Fatty acids C8-10, mixed esters with diPE, isooctanoic acid, PE and triPE (CAS# 189200-42-8), Fatty acids, C16-18 and C18-unsatd., branched and linear ester with trimethylolpropane (CAS# 403507-18-6) and Fatty acids, C5-10, esters with pentaerythritol (CAS # 68424-31-7) were used applying an analogue based read-across approach, in accordance to Regulation (EC) No. 1907/2006 Annex XI, 1.5.

Genetic toxicity (gene mutation) in bacteria in vitro

The mutagenic potential of Fatty acids, C16-18, esters with pentaerythritol (CAS# 85116-93-4) was tested in a Salmonella typhimurium reverse mutation assay comparable to OECD Guideline 471 and under GLP conditions (Banduhn, 1991). The following strains were used: TA 1535, TA 1537, TA 98, TA 100 and TA 1538. Tester strains were incubated with test material concentrations of 8, 40, 200, 1000 and 5000 µg/plate (no toxicity but tested up to precipitating concentrations) in Tween 80 with and without the addition of a metabolic activation system (Aroclor 1254 induced rat liver S9 mix). 2-Aminoanthracene and Sodium Azide, 9-Aminoacridine and 4-Nitro-o-phenylendiamine were used as positive controls with and without S9 mix, respectively.

Positive control materials induced statistically significant increases in the frequency of revertant colonies indicating the satisfactory performance of the test and the activity of the metabolizing system. No increase in the frequency of revertant colonies compared to concurrent negative controls was observed in all strains tested, neither in the presence nor in the absence of metabolic activation. Thus, Fatty acids, C16-18, esters with pentaerythritol did not induce point mutations by base-pair changes or frame-shifts in the genome of the strains tested.

Additionally, the mutagenic potential of Fatty acids, C8-18 and C18-unsatd., esters with trimethylolpropane (CAS# 85186-89-6) was tested in a Salmonella typhimurium reverse mutation assay comparable to OECD Guideline 471 and under GLP conditions (Wiebel, 1999). The following strains were used: TA 1535, TA 1537, TA 98, TA 100 and TA 1538. Tester strains were incubated with test material concentrations of 8, 40, 200, 1000 and 5000 µg/plate (no toxicity but tested up to precipitating concentrations) in acetone with and without the addition of a metabolic activation system (phenobarbitale and beta-naphthoflavone induced rat liver S9 mix). 2-Aminoanthracene and Sodium Azide, 9-Aminoacridine and 4-Nitro-o-phenylendiamine were used as positive controls with and without S9 mix, respectively.

Positive control materials induced statistically significant increases in the frequency of revertant colonies indicating the satisfactory performance of the test and the activity of the metabolizing system. No increase in the frequency of revertant colonies compared to concurrent negative controls was observed in all strains tested, neither in the presence nor in the absence of metabolic activation. No cytotoxicity was observed but the test substance was tested up to limit concentrations. Thus, Fatty acids, C8-18 and C18-unsatd., esters with trimethylolpropane did not induce point mutations by base-pair changes or frame-shifts in the genome of the strains tested.

The mutagenic potential of Fatty acids C8-10, mixed esters with diPE, isooctanoic acid, PE and triPE (CAS# 189200-42-8) was tested in a Salmonella typhimurium reverse mutation assay comparable to OECD Guideline 471 and under GLP (Przygoda, 1995). The following strains were used: TA 1535, TA 1537, TA 98, TA 100 and TA 1538. Tester strains were incubated with test material concentrations of 0, 0.5, 5, 50, 500, 5000 µg/plate in the first experiment and 0, 50, 100, 500, 1000 and 5000 µg/plate in the repeat experiment in acetone with and without the addition of a metabolic activation system (Arochlor 1254 induced rat liver S9 mix). 2-Aminoanthracene and 9-Aminoacridine, N-Methyl-N-Nitro-N-Nitrosoguanidine and 2-nitrofluorene were used as positive controls with and without S9 mix, respectively.

Positive control materials induced statistically significant increases in the frequency of revertant colonies indicating the satisfactory performance of the test and the activity of the metabolizing system. No increase in the frequency of revertant colonies compared to concurrent negative controls was observed in all strains tested, neither in the presence nor in the absence of metabolic activation. No cytotoxicity was observed but Beading of the test substance was observed in the initial assay and the repeat assay at500 µg/plate and higher with and without metabolic activation in all strains. Thus, Fatty acids C8-10, mixed esters with diPE, isooctanoic acid, PE and triPE did not induce point mutations by base-pair changes or frame-shifts in the genome of the strains tested.

A bacterial gene mutation assay (Ames test) was performed with dipentaerythritol ester of nC5/iC9 acids (CAS #647028-25-9) following OECD guideline 471 and under GLP conditions (Thompson, 1992). The tested strains were Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2 uvrA .

The test concentrations for the main study were determined in a preliminary toxicity study with and without metabolic activation at concentrations from 0.15 up to 5000 µg/plate in the strains TA 100 and E. coli WP2 uvrA. The first and second experiment of the main study were performed each in triplicates according to the plate incorporation procedure at concentrations up to 5000 µg/plate (vehicle: acetone) with and without a metabolic activation system (Aroclor 1254-induced rat liver S9-mix). Cytotoxicity was determined by inspection of the bacterial background lawn.

The included positive and negative controls in the experiments showed the expected results and were therefore considered as valid. No increase in the number of revertant colonies was observed in any of the bacterial strains, with and without metabolic activation system. At 5000 µg/plate an oily precipitate was observed in all tested strains. No cytotoxicity was observed up to the highest, precipitating dose.

Thus, the Dipentaerythritol ester of nC5/iC9 acids did not induce mutations in the bacterial mutation tests in the absence and presence of a metabolic activation system in any of the strains tested.

In summary, due to consistently negative results fatty acids, C16-18 and C18-unsatd., hexaesters with dipentaerythritol expected not to be mutagenic to bacteria.

Genetic toxicity (cytogenicity) in mammalian cells in vitro

An in vitro mammalian chromosome aberration test was performed with Fatty acids, C16-18 and C18-unsatd., branched and linear ester with trimethylolpropane (CAS# 403507-18-6) in cultured peripheral human lymphocytes comparable to OECD Guideline 473 and GLP (Durward, 2004). Duplicate cultures of human lymphocytes were evaluated for chromosome aberrations in the presence and absence of metabolic activation (rat liver S9-mix).

In the first experiment cells were exposed for 4 hours to test substance concentrations of 240, 320, 400 µg/mL in acetone with and without metabolic activation. In the second experiment cells were exposed for 4 hours to 240, 320, 400 µg/mL with metabolic activation and for 24 hours to 240, 320, 400 µg/mL followed by 24 hours expression time without metabolic activation.

The test substance did not induce cytotoxicity but a cloudy precipitate was already visible at 40 µg/mL. Mitomycin C and cyclophosphamide were used as positive control substances. Vehicle (solvent) controls induced aberration frequencies within the range expected for normal human lymphocytes. Positive control materials induced statistically significant increases in aberration frequencies indicating the satisfactory performance of the test and of the activity of the metabolizing system. Evaluation of 200 well-spread metaphase cells from each culture for structural chromosomal aberrations revealed no increase in the frequency of chromosome aberrations and polyploid cells at any dose level in comparison to the negative controls. The test material was therefore considered to be non-clastogenic to human lymphocytes in vitro.

Another in vitro mammalian chromosome aberration test was performed with Fatty acids C8-10, mixed esters with diPE, isooctanoic acid, PE and triPE (CAS# 189200-42-8) in Chinese hamster ovary cells (CHO cells) comparable to OECD Guideline 473 and GLP (Przygody, 1995). Duplicate cultures of CHO cells were evaluated for chromosome aberrations in the presence and absence of metabolic activation (rat liver S9-mix).

In the first experiment cells were exposed to the test substance for 3 hours and for 16 hours followed by 16 hours expression time with and without metabolic activation, respectively. Test concentrations used were 40, 80 and 160 µg/mL in acetone. In the second experiment cells were again exposed for 3 hours and for 16 hours followed by 16 hours expression time with and without metabolic activation, respectively. Additionally, cells were exposed for 3 and 16 hours followed by 40 hours expression time with and without metabolic activation, respectively. The same substance concentrations as in experiment I were used.

The test substance did not induce cytotoxicity but a precipitate was visible in the second experiment at 160 µg/mL after 16 hours incubation without metabolic activation. N-Methyl-N-Nitro-N-Nitrosoguanidine and 7,12-Dimethylbenz[a]anthracene were used as positive control substances. Vehicle (solvent) controls induced aberration frequencies within the range expected for normal human lymphocytes. Positive control materials induced statistically significant increases in aberration frequencies indicating the satisfactory performance of the test and of the activity of the metabolizing system. Evaluation of 100 well-spread metaphase cells from each culture for structural chromosomal aberrations revealed no increase in the frequency of chromosome aberrations and polyploid cells at any dose level in comparison to the negative controls. The test material was therefore considered to be non-clastogenic to CHO cells in vitro.

 

An in vitro mammalian chromosome aberration test was conducted with Dipentaerythritol ester of nC5/iC9 acids (CAS # 647028-25-9) in accordance with OECD guideline 473 under GLP conditions (Wright, 2000).

The induction of structural chromosome aberrations was evaluated in human lymphocytes in vitro incubated for 4 h with and without metabolic activation system and 20 h without metabolic activation system (S9-mix from rats treated with Aroclor 1245). Concentrations of 39.06-5000 µg/mL (4 h incubation, with and without S9-mix) and 156.25-5000 µg/mL (20 h incubation, without S9-mix) of the test substance were applied. The vehicle used in the testing was acetone. Cytotoxicity was evaluated calculating the mitotic index of 2000 cells and polyploidy was checked.

There was a cloudy appearance of the test material at all concentrations in both treatment groups after 4 h exposure. The negative and positive controls showed the expected results and were within the range of historical control data. No cytotoxicity was observed up to the highest tested concentration. No increase in the incidence of chromosome aberrations was observed under the conditions of the study.

Thus, the Dipentaerythritol ester of nC5/iC9 acids did not show clastogenic activity in this chromosomal aberration test performed in human lymphocytes in vitro.

 

Since no mutagenic potential was found in the studies above, the Polyol DiPE substance is not considered to have cytogenetic potential in mammalian cells in vitro.

Genetic toxicity (gene mutation) in mammalian cells in vitro

An in vitro Mammalian Cell Gene Mutation Assay according to OECD Guideline 476 and GLP was performed with Fatty acids, C8-18 and C18-unsatd., esters with trimethylolpropane (CAS# 85186-89-6) in mouse lymphoma L5178Y cells (Verspeek-Rip, 2010). The cells were treated for 3 and 24 hours with 8% (v/v) and without S9-mix in the first experiment, respectively and with 12% (v/v) and without S9-mix in the second experiment, respectively. The test substance was tested up to precipitation, the following concentrations were tested: 0.3, 1, 3, 10, 33, 100, 333 and 750 μg/mL. Cyclophosphamide and Methylmethanesulfonate were used as positive controls with and without S9 mix, respectively. No toxicity was observed and all dose levels were evaluated in the absence and presence of S9-mix. Positive and negative controls were valid and in range of historical control data. No significant increase in the mutation frequency at the TK locus was observed after treatment with Fatty acids, C8-18 and C18-unsatd., esters with trimethylolpropane (UVCB) either in the absence or in the presence of S9-mix. It was concluded that Fatty acids, C8-18 and C18-unsatd., esters with trimethylolpropane is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described.

In summary, the substances of the Polyol PE subcategory are not expected to have mutagenic potential in mammalian cells in vitro.

Conclusion for genetic toxicity in vitro

Several read-across studies are available to assess the mutagenic potential in bacteria using analogue based read-across, in accordance to Regulation (EC) No. 1907/2006 Annex XI, 1.5. Since, all studies are negative for genetic toxicity, the Polyol DiPE substance is not considered to induce point mutations by base-pair changes or frame-shifts in the genome of the strains tested. Cytogenicity data and gene mutation data from mammalian cells are available from structural related read across substances. All studies investigating cytogenicity using CHO cells or cultured human lymphocytes are negative for induction of chromosomal aberrations. Equally, no gene mutation effects were found in mammalian cells. Therefore, based on structural related based read-across the Polyol DiPE substance is not considered to have cytogenetic or gene mutation potential in mammalian cells in vitro. 

Genetic toxicity in vivo

Since there are no in vivo genetic toxicity studies available again analogue based read-across, in accordance to Regulation (EC) No. 1907/2006 Annex XI, 1.5, was applied.

An in vivo micronucleus assay with Fatty acids, C5-10, esters with pentaerythritol (CAS # 68424-31-7) was conducted according to OECD guideline 474 and GLP (Griffiths, 1992). The substance was found to be not genotoxic in the micronucleus assay in vivo after intraperitoneal application. A single intraperitoneal injection was given to groups of 5 male and 5 female CD-1 mice at a dose level of 5000 mg/kg bw. Bone marrow samples were taken 24 and 48 hours after dosing.

The positive control induced statistically significant and biologically meaningful increases in micronucleated polychromatic erythrocytes, compared to the vehicle control values, thus demonstrating the sensitivity of the test system to a known clastogen.

Comparison of the percentage of polychromatic erythrocytes showed no significant differences between the female animals treated with the vehicle control or with the test material. A small, but significant decrease was, however, noted in male mice treated with the test material at 5000 mg/kg bw. This small decrease is, however, considered not to be biologically significant compared to the concurrent control values. Therefore, no statistically or biologically significant increases in the incidence of micronucleated polychromatic erythrocytes over the vehicle control values were seen in either sex at either of the sampling times. The substance is found not to be clastogenic in the mouse micronucleus test in vivo.

A second in vivo micronucleus assay with Fatty acids C8-10, mixed esters with diPE, isooctanoic acid, PE and triPE (CAS# 189200-42-8) was conducted according to OECD guideline 474 and GLP (Przygoda, 1995). The substance was found to be not genotoxic in the micronucleus assay in vivo after gavage application. Three oral gavage applications were given to groups of 5 male and 5 female CD-1 mice at a dose levels of 500, 1000 and 2000 mg/kg bw, approximately every 24 hours. Bone marrow samples were taken 24 hours after dosing.

The positive control (cyclophosphamide) induced statistically significant and biologically meaningful increases in micronucleated polychromatic erythrocytes, compared to the vehicle control values thus, demonstrating the sensitivity of the test system to a known clastogen.

Comparison of the percentage of polychromatic erythrocytes showed no significant differences between animals treated with the vehicle control or with the test material. No statistically or biologically significant increases in the incidence of micronucleated polychromatic erythrocytes over the vehicle control values were seen in either sex at either of the concentrations.

Therefore, the substance is found not to be clastogenic in the mouse micronucleus test in vivo.

Conclusion for genetic toxicity in vivo

There are two in vivo micronucleus studies available assessing the cytogenetic potential of analogue read-across substances in vivo. Both assays using Fatty acids, C5-10, esters with pentaerythritol (CAS # 68424-31-7) and Fatty acids C8-10, mixed esters with diPE, isooctanoic acid, PE and triPE (CAS# 189200-42-8) were negative and therefore, the Polyol DiPE substance was considered not to have clastogenic potential in vivo.

 


Justification for selection of genetic toxicity endpoint
Hazard assessment is conducted by means of read-across based on an analogue approach. No study was selected since all available in vitro and in vivo genetic toxicity studies were negative. All available studies are adequate and reliable based on the identified similarities in structure and intrinsic properties between source and target substance and overall quality assessment (refer to the endpoint discussion for further details).

Short description of key information:
Negative results in several Salmonella typhimurium strains , with and without metabolic activation (OECD 471, GLP, analogue approach).
Negative results in mammalian chromosomal aberration test (OECD 473, GLP, analogue approach).
Negative results in mammalian cell gene mutation tests (OECD 476, GLP, analogue approach).
Negative results in mammalian erythrocyte micronucleus test in vivo (OECD 474, GLP, analogue approach).

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on read-across from structurally similar substances, the 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.