Sorbitan, (Z)-9-octadecenoate (2:3)

Endpoint summary

Additional information

Justification for grouping of substances and read-across

The Sorbitan fatty acid esters category covers fatty series of analogous esters comprised of D-glucitol and natural fatty acids. The category contains UVCB substances, which exhibit differences in chain length (C8-C18), degree of esterification (mono-, di-, tri- and higher esters) and extent of unsaturation (saturated and mono unsaturated).

The naming of the substances is in accordance with the European Pharmacopeia (2011).

The available data allows for an accurate hazard and risk assessment of the category and the category concept is applied for the assessment of environmental fate, 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.

 

Genetic toxicity of the Sorbitan fatty acid esters category: 

CAS	EC Name	Genetic toxicity in vitro	Gene mutation in bacteria	Cytogenicity in mammalian cells	Gene mutaion in mammalian cells	Genetic toxicity in vivo	Cytogenicity	Gene mutation
91844-53-0	Sorbitan octanoate (2:3)		negative	--	--		--	--
1338-39-2	Sorbitan laurate		--	negative	negative		--	--
26266-57-9	Sorbitan palmitate		--	--	--
1338-41-6	Sorbitan stearate		negative	ambigous	--
71902-01-7	Sorbitan isooctadecanoate		--	--	--		--	--
8007-43-0	Sorbitan, (Z)-9-octadecenoate (2:3)		negative	--	--		--	--
26658-19-5	Sorbitan tristearate		--	--	--		--	--
26266-58-0	Anhydro-D-glucitol trioleate		--	--	--		--	--
50-70-4	D-glucitol (+)		negative	negative	--		negative negative	--
112-85-6	Docosanoic acid (+)		negative	negative				negative
124-07-2	Octanoic acid (+)		negative

--: no data available

(+): Surrogate substances

 

Genetic toxicity in vitro

Gene mutation in bacteria

In regard to genetic toxicity in vitro, several bacterial mutation assays are available to evaluate the mutagenic potency of Sorbitan fatty acid esters. Specifically, two Ames tests were conducted with Sorbitan stearate including one GLP-guideline performed according to OECD 471 (MHLW Japan 2007). In detail, 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 48h 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 were observed in all tester strains with and without metabolic activation. Validity of the study was confirmed by the respective negative and positive controls.

The second Ames test with sorbitan stearate was performed in the Salmonella typhimurium strains TA 98 and TA 100 (Inoue et al. 1980) which were exposed to 10, 100, 200, 1000 and 2000 µg test substance dissolved in DMSO or water/plate with and without metabolic activation for 48h. No increase in the frequency of revertant mutants was observed in the strains treated with the test substance. Moreover, vehicle, negative and positive controls revealed the expected and valid results. However, due to limited documentation regarding the test system and the results section, this publication is considered as not assignable with reliability of 4.

Further, an additional Ames test was performed with Sorbitan octanoate (2:3) 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.

The above mentioned studies are amended by two Ames tests performed with Sorbitan, (Z)-9-octadecenoate (2:3). One of these studies is a GLP-guideline study conducted equivalent to OECD 471 (Schröder 1998) with the deviation that a tester strain with a primary AT base pair reversion site sensitive for cross-linking agents was not included in the study. In detail, S. typhimurium TA 1535, TA 1538, TA 1537, TA 98 and TA 100 were treated with 8 to 5000 µg test substance/plate dissolved in DMSO for 48 h with and without metabolic activation in a plate incorporation test. The frequency of revertant colonies did not differ between controls and test plates with and without metabolic activation. Cytotoxicity was not observed. The negative and positive controls were valid and revealed the expected results. The second available Ames test was performed by Callander (1995) and 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. However, regarding genetic toxicity the test substance was considered as negative because no increase in the number of revertants was observed.

Overall and taken all the data into consideration, members of the Sorbitan fatty acid esters category did not reveal any sign of mutagenic potency in reverse bacterial mutation assays.

 

Chromosome aberration

In addition to reverse bacterial mutation assays, chromosome aberration tests were performed to determine clastogenic properties of the Sorbitan fatty acid esters. Specifically, one chromosome aberration test was conducted with 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.

In addition, a chromosome aberration test was performed with Sorbitan stearate in chinese hamster lung cells (MHLW Japan 2005). Due to relevant methodological deficiencies, the study was disregarded and not further taken into account for hazard assessment. However, cells were treated with concentrations of 130, 250 and 500 µg/mL without and with 1100, 2200 and 4300 µg/mL with metabolic activation for 6h. In addition, continuous treatment for 24 and 48h at concentrations of 20, 40 and 80 200 µg/mL without metabolic activation was included. Sorbitan stearate induced cytotoxicity as determined by growth inhibition under all applied test conditions. Specifically, growth inhibition of up to 50% was observed at 80 µg/mL without metabolic activation after 48 h and in the 6h treatment period at 500 µg/mL without metabolic activation. Further, growth inhibition of 31% compared to controls was determined after 6 h incubation with 4300 µg/mL with metabolic activation.

After continuous treatment for either 24 or 48 h, the total number of cells with aberrations did not differ significantly between control and exposed cells. Vehicle controls revealed a higher aberration frequency than cells exposed to the low- and mid-dose after 48 h (4/200, 1/200, 2/200 and 4/200 cells with aberration for vehicle, 20, 40 and 80 µg/mL treated cells, respectively).

For the short term exposure of 6 h without metabolic activation, a statistically significant but not dose-dependent increase in the percentage of polyploid cells was determined (0.75%, 4.5%, 7% and 5.5% for vehicle, 130, 250 and 500 µg/mL treated cells, respectively) without inducing a significant increase in the number of aberrant cells (3/200, 0/200, 6/200, 5/200, 3/200 for untreated controls, vehicle, 130, 250 and 500 µg/mL treated cells, respectively). The percentage of polyploid cells in negative and positive control was similar (0.38%) and twofold higher (0.75%) in the vehicle control, indicating no effect of the positive control regarding polyploidy. No effects on polyploidy were observed in the 24 and 48 h treatment periods without metabolic activation.

In contrast, the short term exposure with metabolic activation revealed a dose-dependent decrease in the percentage of polyploidy cells (0.88%, 0.63%, 0.63% and 0.13% for vehicle, 1100, 2200 and 4300 µg/mL treated cells) and an increase in the total number of aberrant cells reaching statistical significance only when gaps are included in the analysis (incl. gaps: 3, 11*, 54* and 93*; excl. gaps: 3, 42, 52, 91 for vehicle, 1100, 2200 and 4300 µg/mL treated cells ). However, according to OECD guideline 473, gaps should not be included in the total aberration frequency.

The validity of the data obtained for the short term exposures was not confirmed as cyclophosphamide (CPA), which requires metabolic activation to induce clastogenic potency, was used as a positive control substance for both approaches, with and without metabolic activation. Further, CPA did not induce a statistically significant increase in the total number of aberrant cells in the presence of metabolic activation system. Therefore, the obtained results for the short term exposure period with and without metabolic activation are inconclusive and can therefore not be taken into account for assessment. Further, no information on sampling and fixation time, metaphase-arresting substance, precipitation and purity of the test substance were given in the report. Regarding to the latter, polyploidy and clastogenicity might possibly be due to impurities with respect to the heterogeneity of mutagenic effects on polyploidy and aberrations. Due to this relevant methodological deficiencies, the study was disregarded and not further taking into hazard consideration.

In summary, two members of the Sorbitan fatty acid ester category were tested in regard to clastogenic potency. Sorbitan laurate did not show any signs of clastogenicity in human lymphocytes and the data on Sorbitan stearate were considered as not reliable and therefore insufficient for hazard assessment.

 

Gene mutation in mammalian cells

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 (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.

 

Genetic toxicity in vivo

The available and reliable regarded in vitro genotoxicity tests did not reveal evidences that members of the Sorbitan fatty acid ester category exhibit mutagenic or clastogenic properties. Thus, in accordance to Regulation (EC) No 1907/2006, no in vivo genotoxicity studies are required to fulfil the data requirements.

Furthermore, members of the Sorbitan fatty acid esters are generally recognised as safe as they are used as food additives to colour, sweeten or preserve foods and are intentionally added to foodstuffs. Moreover, Sorbitan fatty acid esters members are covered by the regulation (EC) 1333/2008 on food additives and are labelled with the following E numbers: E491 sorbitan monostearate, E492 sorbitan tristearate, E493 sorbitan monolaurate, E494 sorbitan monooleate, E495 sorbitan monopalmitate which are listed as colours and anti-foaming agents (please refer to Directive No 95/2/EC, 20.02.1995).

 

Genetic toxicity of breakdown products

All Sorbitans within this category represent esters which are known to hydrolyse into carboxylic acids and alcohols (Müller-Esterl 2004). Therefore, it seems reasonable to suppose that Sorbitans hydrolyse to D-glucitol and fatty acids under physiological conditions as proven for Sorbitan stearate and Anhydro-D-glucitol trioleate in in vitro (Krantz 1951) and in vivo studies (Wick 1953a/b) (see chapter 7.1). Thus, it is feasible to evaluate the probable metabolites, D-glucitol and fatty acids, as read across analogues in regard to genetic toxicity. D-glucitol is included in Annex IV of the Regulation 1907/2006/EC and thus sufficient information is known to consider it as non-hazardous because of intrinsic properties. Thus, D-glucitol is exempted from the registration under the Regulation 1907/2006/EC. Further, D-glucitol is listed in the GRAS register (generally recognised as safe substance) in the United States and is therefore considered to cause minimum risk. In addition, D-glucitol is intentionally used as food additive in order to substitute sugar (Subcommittee on Review of the GRAS list (Phase II) 1972). It is also a naturally occurring substance found in apples, pears, peaches and prunes (Griffin and Lynch 1968, Informatics Inc. 1972).

Moreover, all Sorbitans within this category are esterified with unsaturated C8 to C18 fatty acids or a saturated C18 fatty acid, respectively, which represent naturally occurring substances in either vegetable or animal fat and consequently daily taken up by humans via food. Vegetable and animal fats are listed on Annex V of the REACh regulation and exempted from registration. Further, fatty acids are found as physiological components in the human body. In particular, C16 and C18 fatty acids are necessary for the formation of lipid bilayers of cell membranes (Müller-Esterl 2004).

Genetic toxicity of D-glucitol

However, adverse effects on chromosomes in the anaphase of human embryonic lung cells (WI-38) were investigated in vitro after exposure toD-glucitolat concentrations of 10, 100 and 1000 µg/mL (FDA 1972). In comparison to controls, D-glucitol-treated cells showed moderately dose-dependent increases in the percentage of cells having bridges, the percentage of abnormal cells and the percentage of aberrant cells. Negative and positive controls (Triethylenemelamine, 0.05 µg/mL) were included in the study and considered as valid. However, the reliability of the study was defined as not assignable with RL4 due to limitations in documentation of the test method, the metabolic activation system, evaluation criteria and results.

In addition, two further studies performed by the FDA with 5% D-glucitol (w/v) investigated mutagenicity in reverse mutation assay including the Salmonella typhimurium tester strains G46 and TA 1530 and Saccharomyces cerevisiae D-3 (FDA 1972). Both reports revealed limitations in documentation and are therefore considered as not assignable (RL4). Specifically, no information on the inclusion of a metabolic activation system, evaluation criteria and results were included in the report. However,D-glucitolwas considered as non-mutagenic in regard to mutagenicity in both studies.

In regard to genotoxicity in vivo, only roughly documented data is available (RL4). D-glucitol was tested in a rodent dominant lethal assay in which male rats were either treated orally with 2500, 5000 and 30 000 mg/kg bw in an acute-single dose procedure (treatment once a week over 8 weeks) or in a subacute approach (continuous treatment over 7 weeks) (FDA 1972). A varying number of 16 to 20 animals was used per dose group, depending on the dose and treatment week (no further details given). Positive controls were treated with 0.2 mg/kg bw triethylenemelamine. The average number of implantations, dead implants, corpora lutea and preimplantation loss per pregnant female as well as the number of dead implants per total implants were determined. Sporadic decreases and increases in these reproductive parameters were observed. However, since these effects did not reveal any dose relation and occurred at isolated time points, the findings were considered as fortuitous. Validity of the data was confirmed by the positive control animals. Therefore, the test substance was considered as negative in the rodent dominant lethal test.

In a mammalian bone marrow chromosome aberration test, rats (5/group) were orally treated with 30, 2500 and 5000 mg/kg bwD-glucitoleither for 6, 24, or 48 h or in a subacute approach (FDA 1972). Positive controls were treated with 0.4 mg/kg bw triethylenemelamine. A clearly increased number in the cells with breaks, rearrangements and aberrations was observed in the positive control group, whereas no effects on these indices were seen in the treatment groups. Therefore, the test substance was considered as negative in the conducted in vivo chromosome aberration test.

Furthermore, mutagenic effects of D-glucitol were investigated in a host-mediated assay in an acute and in a subacute approach, measured as mitotic recombination frequency for Saccharomyes cerevisiae (FDA 1972). Mice were orally exposed to different concentrations including a high-dose representing either the maximum tolerated dose or 5 g/kg bw, a low-dose of 30 mg/kg or near the use level, and a medium-dose between the use level and the maximum tolerated dose (dose levels were not further specified). No measurable mutagenic response was observed when mice were treated with the test substance acutely. As only a slight increase in the mitotic recombination frequency was observed in the subacute treatment approach,D-glucitolwas regarded as non-mutagenic in the conducted host-mediated assay.

Genetic toxicity of fatty acids

Sorbitans within this category are all esterified with unsaturated C8 to C18 fatty acids or a saturated C18 fatty acid, respectively. Therefore, docosanoic acid and octanoic acid were chosen as representatives for the fatty acids as probable metabolites besidesD-glucitol. Since docosanoic acid is a C22 fatty acid and ocatanoic acid a C8 fatty acid, they were considered in an interpolation approach to cover the whole range of propable occurring fatty acids after hydrolysis of Sorbitan esters. In vitro data on genotoxicity is available for both substances.

For docosanoic acid, mutagenicity in bacteria was assessed in GLP-study performed according to OECD 471 (Nakajima 2002). Salmonella typhimurium tester strains TA 1535, TA 1537, TA 98, TA 100 and Escherichia coli WP2uvrA were tested. The bacteria were exposed to concentrations of 156, 313, 625, 1250, 2500 and 5000 µg/plate in a pre-incubation assay in the absence and presence of metabolic activation by rat liver S9-mix. Cytotoxicity was not observed. Precipitation was observed in all treatment groups. The test substance is almost insoluble in water (0.016 mg/L) and with DMSO as solvent just a suspension could have been prepared. Therefore, precipitation was not regarded as criterion for exclusion for evaluation. The maximum numbers of revertants observed in the test substance-treated plates were comparable to those in the negative controls with and without metabolic activation in all strains tested. Appropriate positive controls were included into the study design, which gave the expected results.

The clastogenic potential of docosanoic acid in vitro was assessed in a chromosomal aberration test in mammalian cells performed equivalent to OECD 473 under GLP-conditions (Nakajima 2012). The selection of the concentrations used for the main study was based on the cytotoxicity results of a pre-tests and the following concentrations were chosen for the main test: The CHL cells were exposed for 6 h to 578, 1750, 3500 µg/mL with and without metabolic activation, to 350, 700, 1400 and 2800 µg/mL without metabolic activation for 24h and to 288, 575, 1150 and 2300 g/mL for 48 h. Precipitation was observed in the high dose group at continuous exposure. Cytotoxicity was not observed. 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 increases in metaphases with aberrations.

With octanoic acid, an Ames test was performed smiliar as described in OECD 471 (NTP 1985). Salmonella typhimurium tester strains TA 1535, TA 1537, TA 98, TA 100 and TA 97 were treated with 10, 33, 100, 333, 1000 µg/plate with (10% S9-mix) and without metabolic activation as well as with 33, 100, 333, 1000, 3333 µg/plate with (30% S9-mix) and without metabolic activation in a pre-incubation approach. Precipitation was observed at 1000 and 3333 µg/plate in all strains. Cytotoxicity was observed in TA 97 at concentrations of 1000 and 3333 µg/plate without metabolic activation. The appropriate positive controls that were included into the test revealed the expected mutagenicity.

Conclusion for genetic toxicity

In summary, all available and reliable in vitro and in vivo studies conducted with Sorbitan fatty acid esters category members and with the hydrolysis products revealed no effects on genetic toxicity.

A detailed reference list is provided in the technical dossier (see IUCLID, section 13) and within CSR.

Short description of key information:
Negative results when investigated regarding gene mutation in bacteria, mammalian chromosomal aberration, mammalian cell gene mutation tests/DNA damage or repair and with regard to in vivo mammalian cytogenicity

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 Sorbitan fatty acid esters 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 labeled 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.