Registration Dossier

Administrative data

Key value for chemical safety assessment

Additional information

Genetic toxicity

Justification for read-across

There are no data available for Sorbitan C16-18 (even numbered) fatty acid esters, ethoxylated (1-6.5 moles ethoxylated). In accordance with Regulation (EC) No 1907/2006, Annex XI, 1.5 read-across from an appropriate substance is conducted to fulfill the standard information requirements set out in Regulation (EC) No 1907/2006, Annex VII and VIII, 8.4.

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”.

Sorbitan C16-18 (even numbered) fatty acid esters, ethoxylated (1-6.5 moles ethoxylated)represents an UVCB substance composed of polyethoxylated sorbitan esterified mainly with C16 (44%) and C18 saturated fatty acids (54%). The structurally related substance Sorbitan monolaurate, ethoxylated (1-6.5 moles ethoxylated, CAS 9005- 64-5) is also a polyethoxylated sorbitan esterified with lauric acid (C12 saturated fatty acid). Sorbitan stearate (CAS 1338-41-6) and Sorbitan laurate (CAS 1338-39-2) consist of sorbitan esterified either mainly with C16 and C18 fatty acids (sum of C16 and C18 min. 90%) or with C8-18 fatty acids. Due to structural similarities, the presence of common functional groups and the likelihood of common breakdown products, these source substances are considered as structural analogue substances.

Target and source substances are sorbitan esters, which are known to be hydrolysed after oral ingestion at the ester link by pancreatic lipase resulting in the fatty acid moiety and either the polyethoxylated sorbitan or D-glucitol moiety (CIR, 1984; EPA, 2005; Stryer, 1996). Depending on the route of exposure, esterase-catalysed hydrolysis takes place at different places in the organism: After oral ingestion, polysorbates will undergo chemical changes already in the gastro-intestinal fluids as a result of enzymatic hydrolysis. In contrast, substances which are absorbed through the pulmonary alveolar membrane or through the skin enter the systemic circulation directly before entering the liver where hydrolysis will basically take place. 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. The C2 units are cleaved as acyl-CoA, the entry molecule for the citric acid cycle. The alpha- and omega-oxidation, alternative pathways for oxidation, can be found in the liver and the brain, respectively (CIR, 1987). The polyethoxylated sorbitan moiety, is expected to be excreted mostly in the feces and to a minor amount in the urine without further metabolism (CIR, 1984; EPA, 2005). D-glucitol is metabolized to D-glucose or D-fructose (Touster, 1975). D-glucitol will be metabolized by the intestinal microflora (Senti, 1986) or absorbed through the gastrointestinal tract, but slower and less complete than glucose (Allison, 1979). Once absorbed, D-glucitol is primarily metabolized in the liver. The first step involves oxidation by L-iditol dehydrogenase to fructose which is metabolized by the fructose metabolic pathway (Touster, 1975). D-glucitol does not enter tissues other than the liver and does not directly influence the metabolism of endogenous D-glucitol in other tissues (Allison, 1979). Based on the described structural similarities and metabolic fate of target and source substance, 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 in an overall 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 reliable data for the target substance exist, read-across to the analogue substances Sorbitan stearate (CAS 1338-41-6), Sorbitan monolaurate, ethoxylated (1-6.5 moles ethoxylated, CAS 9005-64-5), Sorbitan laurate (CAS 1338-39-2) and Tween 60 (generic CAS 9005-67-8) was conducted.

Genetic toxicity in bacteria (Ames)

CAS 1338-41-6

An Ames tests was conducted with Sorbitan stearate (CAS 1338-41-6) according to OECD 471 and in compliance with GLP (MHLW Japan 2007). A standard battery of Salmonella typhimurium tester strains including TA 98, TA 100, TA 1535 and TA1537 and the E. coli WP2 uvr A pKM 101 were exposed to test substance concentrations of 313, 625, 1250, 2500 and 5000 µg/plate for 48 h with and without metabolic activation in a pre-incubation assay (pre-incubation for 20 min). Precipitation occurred at concentrations of 625 µg/plate and above. No increase in the frequency of revertant colonies compared to concurrent negative controls was observed in all tester strains with and without metabolic activation. Validity of the study was confirmed by the respective negative and positive controls.

 

Genetic toxicity (cytogenicity) in mammalian cells in-vitro

CAS 9005-64-5

The clastogenic potential of Sorbitan monolaurate, ethoxylated (<2.5 EO, Polysorbate 21, CAS 9005-64-5) was assessed in-vitro in a chromosomal aberration test in mammalian cells according to OECD 473 under GLP-conditions (Notox, 2012). The selection of the concentrations used for the main study was based on the results of a pre-test. Based on the findings of cytotoxicity and precipitation, peripheral human lymphocytes were exposed for 3 h to 10, 33 and 100 µg/mL with and without metabolic activation and for 24 h without S9 mix at concentrations of 10, 100 and 300 µg/mL. The harvest time was 24 h after start of exposure. Additionally, cells exposed for 48 h to 10, 100 and 300 µg/mL without S9 mix and for 3 h to 10, 33 and 100 µg/mL with S9-mix and harvested after 48 h were evaluated. Cytotoxicity was observed at 300 µg/mL in the continuous experiment without metabolic activation. There were no biologically and statistically significant increases in numbers of metaphases with aberrations at any exposure duration and at any total culture time, irrespective of metabolic activation. The positive controls resulted in clear and statistically significant increases in metaphases with aberrations.

 

Tween 60 (generic CAS 9005-67-8) 

The clastogenic potential of Tween 60 was tested in a chromosomal aberration test in Chinese hamster cells in a screening test, in which 134 chemicals have been tested (Ishidate and Shigeyoshi, 1977). Cells were treated with 3 different dosages (including a dose which caused 50% growth inhibition, no further details given) over 24 and 48 h. 200 µg/mL. After treatment with Tween 60 for over 48 h, chromosomal aberrations visible as gaps were evident in 1% of cells. In comparison, untreated or vehicle treated cells showed an aberration rate of 1.1 or 0.6%, respectively. Thus, the aberration rate of Tween 60 treated cells lies in the range of untreated and vehicle control cells. Thus, based on the data of this screening study, Tween 60 did not exhibit genetic toxicity in Chinese hamster cells without metabolic activation.

Genetic toxicity (mutagenicity) in mammalian cells in-vitro

CAS 9005-64-5

An in-vitro mammalian cell gene mutation test according to OECD guideline 476 was performed with Sorbitan monolaurate, ethoxylated (<2.5 EO, Polysorbate 21, CAS 9005-64-5) dissolved in ethanol in mouse lymphoma L5178Y cells (Notox, 2012). Cells were treated for 3 h without S9-mix at test substance concentrations of 0.3, 1, 3, 10, 33, 100 and 125 µg/mL and with 8% (v/v) S9-mix at 0.3, 1, 3, 10, 33, 100 and 300 µg/mL. Further, exposure for 24 h occured without S9-mix at 0.3, 3, 10, 33, 100, 150 and 190 µg/mL as well as with 12% (v/v) S9-mix at 0.3, 3, 10, 33, 100, 200, 300 and 350 µg/mL. Precipitation was observed at concentrations of 100 µg/mL and above. The mutation frequency was not increased in any of the treatment groups and no cytotoxicity was observed. Appropriate positive and negative controls were included in the experiment and revealed the expected results thereby validating the study.

CAS 1338-39-2

An in-vitro mammalian cell gene mutation test according to OECD guideline 476 was performed with Sorbitan laurate (CAs 1338-39-2) 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 (10, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 300 and 333 µg/mL) and for 3 h in the presence of 8% (v/v) S9-mix at concentrations of 10 – 350 µg/mL (10, 33, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325 and 350 µg/mL) or 12% % (v/v) S9-mix at 10 – 275 µ/mL (10,25, 50, 75, 100, 125, 150, 175, 200, 225, 250 and 275 µg/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 (0.1, 1, 10, 25, 50, 75, 100, 125, 150, 175, 200 and 225 µg/mL). 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.

Conclusion on genetic toxicity

The available data do not provide evidence that the source substances exhibit mutagenic or clastogenic properties in bacteria or mammalian cells. Therefore, based on common functional groups and structural similarities, no properties for genetic toxicity are expected for Sorbitan C16-18 (even numbered) fatty acid esters, ethoxylated (1-6.5 moles ethoxylated).

References:

Allison, R.G. (1979). Dietary sugars in health and disease III. D-glucitol. Contract No. 223-75-2090, Bureau of foods, Food and Drug Administration, Dept. of Health and Human Services, Washington, D.C. 20204, USA

CIR (1984). Final report on the safety assessment of polysorbat 20, 21, 40, 60, 61, 65, 80, 81 and 85. Journal of the American College of Toxicology, 3(5): 1- 82

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

EPA (2005). ACTION MEMORANDUM. Reassessment of six inert ingredient exemptions from the requirement of a tolerance. United States Environmental Protection Agency, Washington, D.C. 20460, USA

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, D.C. 20204, USA

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

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


Justification for selection of genetic toxicity endpoint
Hazard assessment is conducted by means of a read-across from structural analogues. All available in vitro genetic toxicity studies were negative. Furthermore, 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): CAS 1338-416: negative with and without metabolic activation in S. typhimurium TA 98, TA 100, TA 1535 and TA 1537 and E. coli WP2 uvr A pkm 101
Chromosome aberration (OECD 473): CAS 9005-64-5: negative in human lymphocytes with and without metabolic activation; Tween 60 (generic CAS 9005-67-8): negative in Chinese hamster cells without metabolic activation
Gene mutation (OECD 476): CAS 9005-64-5 & 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

Based on read-across, 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.