Registration Dossier

Administrative data

Key value for chemical safety assessment

Additional information

Genetic toxicity

Justification for read-across

There are no data for genetic toxicity available for Fatty acids, rape-oil, mixed esters with 1,4:3,6-dianhydro-d-glucitol, sorbitan and sorbitol. In accordance with Regulation (EC) No 1907/2006, Annex XI, 1.5 read-across from appropriate substances is conducted to fulfill the standard information requirements set out in Regulation (EC) No 1907/2006, Annex VIII, 8.5.

According to 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) “to avoid the need to test every substance for every endpoint”. 

 

Fatty acids, rape-oil, mixed esters with 1,4:3,6-dianhydro-d-glucitol, sorbitan and sorbitol represents an UVCB substance comprised of different Sorbitan fatty acid ester, mainly of mono-, di- and tri-esters of sorbitol, sorbitan and 1,4:3,6-dianhydro-d-glucitol esterified with natural fatty acids with a chain length ranging from of C16 – C20, mostly C18 mono-unsaturated.

 

Sorbitan fatty acid esters are known to be stepwise hydrolysed to the respective fatty acid and the alcohol moiety, which will be present mostly as the open chain isomer D-glucitol depending on the pH (Stryer, 1996). The first cleavage product, the fatty acid, is stepwise degraded by beta-oxidation based on enzymatic removal of C2 units in the matrix of the mitochondria in most vertebrate tissues. For the complete catabolism of unsaturated fatty acids such as oleic acid, an additional isomerization reaction step is required. The alpha- and omega-oxidation, alternative pathways for oxidation, can be found in the liver and the brain, respectively (CIR, 1987). The alcohol residue, mostly D-glucitol, is absorbed from the gastro-intestinal tract and can be metabolized by the intestinal microflora (Senti, 1986) or in the liver (Touster, 1975). Based on the common metabolic fate of Sorbitan fatty acid esters, the read-across approach is based on the presence of common functional groups, common precursors and the likelihood of common breakdown products via biological processes, which result in structurally similar chemicals and hence exhibit similar toxicokinetic behaviour. For further details on the read-across approach, please refer to the analogue justification in section 13 of the technical dossier.

 

As no data are available on genetic toxicity of Fatty acids, rape-oil, mixed esters with 1,4:3,6-dianhydro-d-glucitol, sorbitan and sorbitol , read-across to reliable data on the analogue substances Sorbitan octanoate (2:3) (CAS 91844-53-0), Sorbitan, (Z)-9-octadecenoate (2:3) (CAS 8007-43-0) and Sorbitan laurate (CAS 1338-39-2) was conducted.

 

Gene mutation in bacteria

CAS 91844-53-0 and CAS 8007-43 -0

In regard to genetic toxicity in vitro, bacterial mutation assays are available for the read-across analogue substances Sorbitan octanoate (2:3) and Sorbitan, (Z)-9-octadecenoate (2:3).

Mutagenic properties of Sorbitan octanoate (2:3) were tested in an bacterial reverse mutation test according to OECD 471 under GLP conditions (Sokolowski 2006). Salmonella typhimurium tester strains TA 98, TA 100, TA 1535, TA 1537 and TA 102 were treated with 3 to 5000 µg test substance/plate dissolved in DMSO for 48 hours with or without S9-mix in a plate pre-incubation test (pre-incubation for 1 h). The concentrations were chosen based on a range-finding study. No increase in the frequency of revertant colonies compared to concurrent negative controls was observed in all tester strains with and without metabolic activation. The negative and positive controls confirmed validity of the conducted study. Thus, the structural analogue substance Sorbitan octanoate (2:3) did not exhibit mutagenic properties in reverse bacterial mutation assays.

 

Furthermore, Sorbitan , (Z)-9-octadecenoate (2:3) was tested in a second Ames test performed by Callander (1995) which included Salmonella typhimurium tester strains TA 98 and TA 100. Due to limited documentation of concentrations, test system, cytotoxicity, controls and results the quality of the publication was assessed with a Klimisch score of 4.

 

Chromosome aberration

CAS 1338-39-2

In addition to reverse bacterial mutation assays, a chromosome aberration test is available for Sorbitan laurate according to OECD 473 under GLP conditions with cultured peripheral human lymphocytes (Buskens 2010). Cells were treated with the test substance in presence and absence of a metabolic activation system in duplicates at concentrations of 33 to 333 µg/mL 3h, at 50 to 600 µg/mL and at 100 to 500 µg/mL for 3, 24 and 48h, respectively. These concentrations were determined in range-finding studies based on precipitation and cytotoxicity. In the main experiment, no cytotoxicity was observed up to precipitating concentrations starting at 333 µg/mL. The test substance did not show clastogenic potency at all concentrations and treatment periods tested. Validity and reliability of the study was confirmed by the respective positive controls which induced significant chromosome aberrations and polyploidy in the applied test system.

 

Gene mutation in mammalian cells

CAS 1338-39-2

An in vitro mammalian cell gene mutation test according to OECD guideline 476 was performed with Sorbitan laurate dissolved in DMSO in mouse lymphoma L5178Y cells (Verspeek-Rip, 2010). Cells were treated for 3 h without metabolic activation at several test substance concentrations ranging from 10 – 333 µg/mL and for 3 h in the presence of 8% (v/v) S9-mix at concentrations of 10 – 350 µg/mL or 12% % (v/v) S9-mix at 10 – 275 µ/mL. In addition, a longer incubation period of 24 h without supplementation of S9 mix was included in which test concentrations of 0.1 – 225 µg/mL were applied. Precipitation of the test substance was observed at concentrations of 333 µg/mL. Cytotoxicity was observed starting at 150 µg/mL without S9-mix and at 175 µg/mL with S9-mix. In contrast to the positive control, the mutation frequency was not increased in any of the treatment groups. Positive and negative controls were valid and lay within the range of historical data. Thus, Sorbitan laurate did not induce gene mutations in mouse lymphoma L5178Y cells.

 

Overall conclusion for genetic toxicity

In summary, the available and reliable genotoxicity studies conducted with the read-across substances Sorbitan octanoate (2:3), Sorbitan laurate and Sorbitan, (Z)-9-octadecenoate (2:3) revealed neither mutagenic nor clastogenic properties. Therefore, Fatty acids, rape-oil, mixed esters with 1,4:3,6-dianhydro-d-glucitol, sorbitan and sorbitol is not expected to exhibit genotoxic properties.

 

References

CIR (1987). Final report on the safety assessment of oleic acid, lauric acid, palmitic acid, myristic acid, stearic acid. J. of the Am. Coll. of Toxicol.6 (3): 321-401

 

Senti, F.R. 1986. Health aspects of sugar alcohols and lactose. Contract No. 223-83-2020, Center for food safety and applied nutrition, Food and Drug Administration, Dept. of Health and Human Services, Washington, DC 20204, USA

 

Stryer, L. 1996. Biochemie. Spektrum Akademischer Verlag; Auflage: 4th edition

Suldano, S., Gramenzi, F., Cirianni, M., Vittozzi, L. (1992): Xenobiotic-metabolizing enzyme systems in test fish - IV. Comparative studies of liver microsomal and cytosolic hydrolases. Comparative Biochemistry and Physiology Part C: Comparative Pharmacology. 101(1), 117-123.

 

Touster, O. 1975: Metabolism and physiological effects of polyols (alditols). In : Physiological effects of food carbohydrates.Washington, DC: American Chemical Society. p 229-239

 


Justification for selection of genetic toxicity endpoint
Hazard assessment is conducted by means of a read-across based on a read-across from structural analogues. All available studies are adequate and reliable based on the identified similarities in structure and intrinsic properties between source and target substances and overall quality assessment (refer to the endpoint discussion for further details).

Short description of key information:
Genetic toxicity in vitro:
Gene mutation (OECD 471): negative with and without metabolic activation in S. typhimurium TA 98, TA 100, TA 102, TA 1535 and TA 1537
Chromosome aberration (OECD 473): negative in human lymphocytes with and without metabolic activation
Gene mutation (OECD 476): CAS 1338-39-2: negative in L5178Y mouse lymphoma cells with and without metabolic activation

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The available data on genetic toxicity of structural analogues do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.