Dehydrated sorbitol, C18 (unsaturated) fatty acid esters, ethoxylated

Endpoint summary

Additional information

Genetic toxicity

Justification for grouping of substances and read-across

There are no data available on the genetic toxicity of Sorbitan monooleate, ethoxylated (1-6.5 moles ethoxylated; CAS 9005-65-6). In order to fulfil the standard information requirements set out in Annex VIII, 8.5., in accordance with Annex XI, 1.5., of Regulation (EC) No 1907/2006, read-across from structurally related substances Tween 80 (CAS 9005-65-6 polysorbat 80 [sorbitan monooleate ethoxylated (20EO)]) and sorbitan monolaurate, ethoxylated (CAS 9005-64-5, poylsorbat 21) 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).

The test substance Sorbitan monooleate, ethoxylated (1-6.5 moles ethoxylated) represents an UVCB substance composed of polyethoxylated sorbitan esterified mainly with C18 unsaturated fatty acids (>60 - 100%). The structurally related substances Tween 80 (CAS 9005-65-6 polysorbat 80 [sorbitan monooleate ethoxylated (20EO)]) and sorbitan monolaurate, ethxylated (polysorbat 21, CAS 9005-64-5) are considered as structural analogue substance due to structural similarities, the presence of common functional groups and the likelihood of common breakdown products: Sorbitan monooleate, ethoxylated (1-6.5 moles ethoxylated), Tween 80 (CAS 9005-65-6 polysorbat 80 [sorbitan monooleate ethoxylated (20EO)]) and sorbitan monolaurate, ethoxylated (poylsorbat 21) are polyethoxylated sorbitan esters linked to oleate or laurate, with ethoxylation degrees of 3 – 5, 20 or 1 -7 EO respectively.

Target and source substances are polyethoxylated sorbitan esters, also called polysorbates, which are known to be hydrolysed after oral ingestion at the ester link by pancreatic lipase resulting in the fatty acid moiety and the polyethoxylated sorbitan moiety (CIR, 1984; EPA, 2005; Fruijitier-Pölloth, 2005). 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. 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 second cleavage product, 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; Fruijitier-Pölloth, 2005). Based on the common metabolic fate of polyethoxylated 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.

 

 

Discussion

Genetic toxicity in vitro

Gene mutation in bacteria in vitro

CAS 9005-65-6 (Tween 80 (CAS 9005-65-6 polysorbat 80 [sorbitan monooleate ethoxylated (20EO)]) )

The potential mutagenicity of Tween 80 was assessed in bacteria in an Ames test similar to OECD Guideline No. 471 (NTP, 1992). In two independent experiments, S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 were exposed to concentrations of 100, 333, 1000, 3333 and 10000 µg/plate in the presence and absence of metabolic activation (S9 mix), respectively. Furthermore, a repeat experiment with the test substance was performed in the absence of S9 mix. No cytotoxicity was noted in the treated strains when compared to controls in any of the experiments performed. In the absence of S9 mix, the test substance resulted in a weekly positive result in TA 98 in the second experiment at concentrations of 333, 1000 and 3333 µg/plate. However, no positive results were obtained after treatment with any concentration in the first experiment, and also the repeat experiment with the test substance in the absence of S9 mix could not confirm the positive result of the second trial. Moreover, no positive results were observed in any of the other test strains, neither in the presence nor in the absence of metabolic activation. The positive and negative control substances for each tester strain showed the expected results, thereby confirming the validity of the study. Under conditions of this assay, the test substance was found to be non-mutagenic in S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, both in the absence of presence of S9-mix.

CAS 9005-64-5

Mutagenicity in bacteria was assessed in a GLP-study performed according to OECD 471 with sorbitan monolaurate, ethoxylated (polysorbat 21) (Verspeek-Rip, 2012). Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and Escherichia coli WP2 uvrA were tested. A range-finder test served for the determination of the test concentrations and was considered as a pre-test for toxicity. In the main tests, bacteria were exposed to concentrations of 33, 100, 333, 1000, 3330 and 5000 µg/plate in the plate incorporation assay in the absence and presence of S9 mix containing 5 or 10% rat liver S9. Cytotoxicity was observed in strains TA 1535 and TA 100 at 3330 µg/plate and above and in TA1537 at 5000 µg/plate without metabolic activation. Further, with metabolic activation, cytotoxicity was observed in TA 1535 at 5000 µg/plate, in TA 1537 at 3330 µg/plate and above and in TA 100 at 333, 1000, 3330, 5000 µg/plate. Precipitation of the test substance was not observed. The maximum numbers of revertants observed in the test substance-treated plates were comparable to those of the negative controls for all strains tested, irrespective of metabolic activation. Appropriate positive controls were included into the study design, which gave the expected results and validated the study. Based on the results of this study, the test substance was considered to be not mutagenic in Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and Escherichia coli WP2 uvrA, bothin the absence of presence of S9-mix.

 

Cytogenicity in vitro

CAS 9005-65-6 (Tween 80 (CAS 9005-65-6 polysorbat 80 [sorbitan monooleate ethoxylated (20EO)]))

A chromosome aberration test was performed with Tween 80 in Chinese Hamster Ovary CHO-W-B1 cells (NIEHS, 1986), which, however, was not taken into account for hazard assessment due to relevant methodological deficiencies. In this study, cells were treated with concentrations of 300, 500, 1000 and 1600 µg/mL in the absence of metabolic activation for a period of 8-12 h, whereas cells were exposed for 2 h at concentrations of 100, 160, 300 500, 750 and 1000 µg/mL in the presence of metabolic activation.

In the short-term exposure experiment with metabolic activation, a linear trend for the increase in the percentage of cells with chromosomal aberrations including "simple" (breaks and terminal deletions), "complex" (rearrangements and translocations), and "other" (pulverised cells, despiralised chromosomes, and cells containing 10 or more aberrations) was determined (2.0%, 1.5%, 3.0%, 4.0%, 5.0% and 13.5% for concentrations of 100, 160, 300 500, 750 and 1000 µg/mL, respectively, compared to 0.75% in controls. Based on the positive result, a repeat experiment in the presence of S9 mix with concentrations of 160, 300, 500 and 1000 µg/mL was performed. However, this experiment failed to reproduce the positive results of the first experiment as the percentage of total chromosomal aberrations compared to controls was only increased at 160 and 500 µg/mL, but not at 300 µg/mL. Due to unknown reasons, the highest concentration of the repeat experiment was not analysed for chromosomal aberrations. As no dose-dependency was observed, only a week evidence for a positive result in the presence of S9 mix was determined that remained to be further elucidated. Although the validity of the data obtained for the short term exposures was confirmed by suitable positive control substance in the presence and absence of metabolic activation, the obtained results for the short term exposure period with and without metabolic activation remained inconclusive due to lacking verification experiments. Furthermore, no detailed information on different types of structural chromosome aberrations, possible precipitation and cytotoxicity effects as well as no details on the purity of the test substance were given in the report. Regarding to the latter, clastogenicity might possibly be due to impurities with respect to the observed heterogeneity of effects on chromosomal aberrations. Due to this relevant methodological deficiencies, the study was disregarded and not further taking into hazard consideration.

Further data on cytogenetic effects of Tween 80 are provided from the results of a sister chromatid exchange (SCE) assay in Chinese Hamster Ovary CHO-W-B1 cells (NIEHS, 1986). Cells were treated with concentrations of 16, 50, 160 and 500 µg/mL in the presence and absence of metabolic activation for a period of 2 h and 26 h, respectively. Two hours after initial exposure, cells were simultaneously treated with bromodesoxyuridine for 24 h to label sister chromatids for the detection of SCEs. After cell harvest and staining, 50 second-division metaphase cells were scored to determine the frequency of SCE/cell for each treatment concentration. An SCE frequency 20% above the concurrent solvent control value was chosen as a statistically conservative positive response. The results of this study showed that no increase in the frequency of SCE in any of the treated cells over controls was apparent. The included positive and solvent controls showed the expected result and thus verified the sensitivity of the assay. Thus, under the conditions of this study, no reciprocal exchanges of DNA between two sister chromatids of a duplicating chromosome, and thus no adverse effects on the chromosome of Chinese Hamster Ovary CHO-W-B1 cells were observed.

CAS 9005-64-5

The clastogenic potential of the test substance in-vitro was assessed in a chromosomal aberration test in mammalian cells according to OECD 473 under GLP-conditions (Verbaan, 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 were 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. Cytotoxicity was observed at 300 µg/mL in the continuous experiments (24 and 48 exposure time) 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. Overall, the available data on the substance show that it is not clastogenic in peripheral human lymphocytes.

Gene mutation 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 (polysorbat 21, 1 - 7 EO) dissolved in ethanol in mouse lymphoma L5178Y cells (Verspeek-Rip, 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 concentrations of 0.3, 1, 3, 10, 33, 100 and 300 µg/mL. Further, exposure for 24 h was performed without S9-mix at concentrations of 0.3, 3, 10, 33, 100, 150 and 190 µg/mL as well as with 12% (v/v) S9-mix at concentrations 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. Based on the results of this study, the substance was not considered to be mutagenic in mouse lymphoma L5178Y cells.

 

Genetic toxicity in vivo

CAS 9005-65-6 (Tween 80 (CAS 9005-65-6 polysorbat 80 [sorbitan monooleate ethoxylated (20EO)]))

A Mammalian Erythrocyte Micronucleus Test with Tween 80 was performed in male CBA mice (Jenssen and Ramel, 1978 and 1980). Groups of 3 animals either received a single dose of the test substance at 75 mg/kg bw or the vehicle alone via oral gavage. After a post-exposure period of 30 h, femoral bone marrow was taken from treated and control animals to determine the frequency of micronucleated polychromatic erythrocytes. No increases in the frequency of micronuclei in polychromatic erythrocytes of the femoral bone marrow of male CBA mice exposed to 75 mg/kg bw were observed compared to controls. Several substances known to induce chromosomal aberrations in vivo were tested in parallel to the Tween 80 and were found to significantly increase the frequency of polychromatic erythrocytes with micronuclei compared to controls. Under the conditions of this experiment, the test substance was negative in the Mammalian Erythrocyte Micronucleus Test in male CBA mice.

 

Conclusions for genetic toxicity

There are no data available on the genotoxicity of Sorbitan monooleate, ethoxylated (1-6.5 moles ethoxylated). However, read-across from the structurally related substance sorbitan monolaurate, ethoxylated (1 - 7 EO, polysorbat 21, CAS9005-64-5)showed that the results of genetic toxicity studies performed in bacteria and mammalian cells with and without metabolic activation were consistently negative (Verspeek-Rip, 2012; Verbaan, 2012; Verspeek-Rip, 2012). In addition, negative results for mutagenicity in bacteria and cytogenicity were obtained from an Ames test and sister chromatid exchange assay, respectively, with the structurally related Tween 80 (CAS 9005-65-6 polysorbat 80 [sorbitan monooleate ethoxylated (20EO)]) (NTP, 1992; NIEHS, 1986). Although a week positive result for clastogenicity was observed in an in vitro chromosome aberration test with Tween 80 in the presence of S9 mix, these results were not considered reliable due to relevant methodological deficiencies, and were therefore not taken into account for hazard assessment. Furthermore, data from a Micronucleus test in mice did not demonstrate a clastogenic potential of Tween 80 in vivo.

Based on the available data, Sorbitan monooleate, ethoxylated (1-6.5 moles ethoxylated) is not anticipated to have a genotoxic potential.

 

References (not included in IUCLID):

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 Protetio Agency, ashington, D.C. 20460

 

Fruijitier-Pölloth (2005). Safety assessment on polyethylene glycols (PEGs) and their derivatives as used in cosmetic products.Toxicology 214, 1 - 38

 

Justification for selection of genetic toxicity endpoint
Hazard assessment is conducted by means of a read-across from a structural analogues. All available in vitro and in vivo 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 9005-65-6 (polysorbat 80 [sorbitan monooleate ethoxylated (20EO)]; Tween 80 ): negative with and without metabolic activation in S. typhimurium TA 98, TA 100, TA 1535 and TA 1537
Gene mutation (OECD 471): CAS 9005-64-5 (sorbitan monolaurate, ethoxylated; polysorbat 21 [1 - 7EO]): negative with and without metabolic activation in S. typhimurium TA 98, TA 100, TA 1535 and TA 1537 and E. coli WP2 uvrA
Chromosome aberration (SCE): CAS 9005-65-6 (polysorbat 80 [sorbitan monooleate ethoxylated (20EO)]; Tween 80): negative in chinese hamster cells CHO-W-B1 with and without metabolic activation
Chromosome aberration (OECD 473): CAS 9005-64-5 (sorbitan monolaurate, ethoxylated; polysorbat 21 [1 - 7EO]): negative in human lymphocytes with and without metabolic activation
Gene mutation (OECD 476): CAS 9005-64-5 (sorbitan monolaurate, ethoxylated; polysorbat 21 [1 - 7EO]): negative in L5178Y mouse lymphoma cells with and without metabolic activation

Genetic toxicity in vivo:
Cytogenicity (MNT): CAS 9005-65-6 (polysorbat 80 [sorbitan monooleate ethoxylated (20EO)]; Tween 80) : negative in CBA mice (male)

Endpoint Conclusion: No adverse effect observed (negative)