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

Endpoint summary

Description of key information

Repeated dose toxicity, oral: the toxicity studies for CAS 9005-65-6 (Sorbitan monooleate, ethoxylated; Tween 81 [5EO] and polysorbat 80 [sorbitan monooleate ethoxylated (20EO)]; Tween 80) revealed NOAELs ≥ 1000 and a LOAEL ≥ 2000 mg/kg mg/kg bw/day  

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Quality of whole database:

The available information comprises insufficient data for assessment from the substance itself and adequate and reliable (Klimisch score 2) studies from a reference substance with similar structure and intrinsic properties. Read-across is justified based on common functional group(s), common precursors/breakdown products and similarities in PC/ECO/TOX properties (refer to endpoint discussion for further details).

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated dose toxicity

Justification for grouping of substances and read-across

There are only limited data available on the oral repeated dose 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 IX, 8.6.2., in accordance with Annex XI, 1.5., of Regulation (EC) No 1907/2006, read-across from the structurally related substance Tween 80 (CAS 9005-65-6 polysorbat 80 [sorbitan monooleate ethoxylated (20EO)]) 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 substance Tween 80 (CAS 9005-65-6 polysorbat 80 [sorbitan monooleate ethoxylated (20EO)]) is 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) and Tween 80 (CAS 9005-65-6 polysorbat 80 [sorbitan monooleate ethoxylated (20EO)]) are polyethoxylated sorbitan esters linked to oleate with ethoxylation degrees of 3 – 5 or 20 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

Repeated dose toxicity, oral

Subacute

CAS 9005-65-6 (Sorbitan monooleate, ethoxylated (Tween 81 [5EO])

In a 6-week subacute feeding study, male rats were administered Tween 81 at a dietary concentration of 1 and 4% in diet, corresponding to dose levels of ca. 2000 and 8000 mg/kg bw/day, respectively (Krantz, 1945). Groups of 12 animals received the test substance in the diet, whereas a further group of 10 animals was administered the plain diet and served as controls. No data on mortality and clinical signs were reported. There was no difference in body weight changes between the treated and control animals. No changes in haematological parameters (only WBC and RBC were determined) were observed in treated animals compared to controls.

Histopathological examination of the livers, kidneys, intestines and bladders of each 3 animals of the control and test groups was performed. As no dose-related effects were observed and/or effects were also observed in control animals, the reported lesions in kidney and liver at both dietary concentrations were not considered to be treatment-related.

Based on these results, a NOAEL ≥ 8000 mg/kg bw/day was derived for male rats.

 

Subchronic

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

The subchronic oral toxicity of Tween 80 was investigated in male and female F344/N rats in a GLP-conform study similar to OECD guideline 408 (NTP, 1992). The test substance was administered daily ad libitum for a period of 13 weeks to groups of 10 animals per sex at dietary concentrations of 3000, 6000, 12500 and 50000 ppm, corresponding to doses of 310, 509, 1020, 2075 and 4132 mg/kg bw/day in males and 384, 765, 1567, 3096 and 6211 mg/kg bw/day in females, respectively. A similar constituted control group received the plain diet. During the study period, no mortalities and no clinical signs were observed. No changes in mean body weights and body weight gain as well as food consumption were observed in treated animals of all dose groups compared to controls. Statistically significant changes in absolute and/or relative organ weights, which occasionally, but not dose-related occurred in liver, lungs and thymus of treated males as well as in heart of treated females, were not considered biologically significant. Macroscopic and microscopic examinations did not reveal any substance-related effects in animals of all dose groups. Based on results of this study, a NOAEL of ≥ 4132 and 6211 mg/kg bw/day for male and female Fischer 344 rats, respectively, was derived.

 

Chronic

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

The oral chronic toxicity of Tween 80 was investigated in a GLP-conform carcinogenicity study in F344/N rats (NTP, 1992). Sixty animals per sex and test group received the test substance ad libitum at dietary concentrations of 25000 and 50000 ppm, corresponding to doses of 1174 and 2415 mg/kg bw/day in males and 1344 and 2745 mg/kg bw/day in females, respectively. A further group of sixty animals per sex received the plain diet and served as controls. All animals were treated for a period of 103 weeks, except for 10 animals per sex of each control and treatment group which were already sacrificed after 15 months for interim examinations (determination of organ weight as well as gross pathology and histopathology).

Overall, no treatment-related effects on survival, no clinical signs of toxicity, no changes in body weight, body weight gain, food consumption and organ weights of brain, right kidney and liver (only determined at the 15-months interim sacrifice) as well as no treatment-related findings at gross pathology and histopathology (neoplastic and non-neoplastic lesions) were observed in females at both dietary dose levels as well as in males at the lower dietary concentration of 25000 ppm when compared to controls.

No treatment-related gross pathological findings were observed in treated animals of all dose levels at the 15-month interim sacrifice and at the end of the 2-year study period.

Although the terminal survival (in percent) of treated male rats of both dose groups was lower than that of the control group, the mean survival (in days) in treated male rats was similar to that of the controls up to Week 93 of the study. Therefore, the decreased terminal survival in treated males was not considered to affect the evaluation of the carcinogenic potential of the test substance in the male rats, and was thus not regarded to be treatment-related.

Histopathological examination of males receiving dietary concentration of 50000 ppm revealed a slight increase in the incidence of benign (+56% compared to controls) pheochromocytoma in adrenal medulla and an increased total incidence of pheochromocytoma in adrenal medulla of males (benign and malignant: +58% compared to controls). The incidence of tumours in the high-dose group (58%) in this study exceeded the historical control range of 22% to 48% for male rats. Nearly all of the pheochromocytoma in all groups occurred in a single gland. The incidences of bilateral neoplasms (4/50, 4/50, 7/50) and malignant neoplasms were low and were similar across treatment groups. Thus, the increased incidence of pheochromocytoma in high-dose males was due to an increase in the number of benign pheochromocytoma occurring in a single gland. In an evaluation of historical control incidences in male F344/N rats based upon a broader range of NTP studies than those included in the recent historical control data, Haseman et al. (1990) found incidences of pheochromocytoma in untreated male rats as high as 65%. Thus, the slight increase in incidence of pheochromocytoma in the high-dose male rat group was judged to be of questionable significance. In addition, however, there was also an increase in the incidence of adrenal medulla hyperplasia, a lesion generally considered to be the precursor to pheochromocytoma, in the low-dose group, but not in the high-dose group. The marginal increased incidence of pheochromocytoma in combination with the lower incidence of hyperplasia in the high dose group compared to the low-dose group was considered to be an equivocal finding. Furthermore, there was no effect in the adrenal glands of female rats. Due to the lack of mechanistic studies, the relationship between the increase in proliferative adrenal medulla lesions in male rats in the present study and the test substance was uncertain.

At the end of the 2-year study period, further statistically significant changes in the incidences of neoplastic and non-neoplastic lesions were observed in the haematopoietic system and in spleen, which, however, were incidental or of low incidence, and thus not considered to be treatment-related.

Based on the equivocal finding of carcinogenic activity at the highest dietary concentration, the NOAEL for male Fischer 344/N rats was set at 25000 pm, corresponding to 1174 mg/kg bw/day. In females, the NOAEL was considered to be ≥ 50000 ppm, which equivalent to dose levels ≥ 2745 mg/kg bw/day.

A similarly performed GLP-conform oral chronic toxicity study with Tween 80 is available in B6C3F1 mice (NTP, 1992). Sixty animals per sex and test group received the test substance ad libitum at dietary concentrations of 25000 and 50000 ppm, corresponding to doses of 5003 and 10811mg/kg bw/day in males and 6487 and 13197 mg/kg bw/day in females, respectively. A further group of sixty animals per sex received the plain diet and served as controls. All animals were treated for a period of 103 weeks, except for 7-10 animals per sex of each control and treatment group which were already sacrificed after 15 months for interim examinations (determination of organ weight as well as gross pathology and histopathology).

No treatment-related effects on survival, no clinical signs of toxicity, no changes in food consumption and organ weights of brain, right kidney and liver (only determined at the 15-months interim sacrifice) as well as no treatment-related gross pathological findings were observed in males and females at 25000 and 50000 ppm.

In females of the high dose group (50000 ppm), a slight reduction (-11%) in final mean body weights was observed compared to controls, whereas no alterations in body weight were noted in males of the same dose group and in females of the lower dose group.

During the 15-month interim evaluations, compound-related histopathological lesions occurred in 5 of 10 females from the high dose group. These lesions included mild hyperplasia of the forestomach epithelium and inflammation of the forestomach. No test substance-related non-neoplastic lesions were observed in treated males of both dose groups.

At the end of the 2-year study period, compound-related lesions of the forestomach (hyperplasia of the epithelium, inflammation, and ulcers) occurred in mice of all dose groups (including controls). However, the incidences and severity of hyperplasia of the forestomach epithelium (hyperplasia, squamous) and inflammation of the forestomach were significantly increased in males and females that received the highest concentration in the diet (50000 ppm). Furthermore, a significantly increased incidence of ulcers was present in the forestomach of females, but not males, from the high dose group. In several animals of the high dose group, chronic active inflammation, a scattering of small to moderate number of mixed Iymphocytes, macrophages, and neutrophils, often accompanied by varying amounts of fibrosis, were present in the forestomach wall beneath areas of hyperplasia.

Based on the treatment-related effects on the forestomach at the highest dietary concentration, the NOAEL for male and female mice was considered to be 25000 ppm, corresponding to 5003 and 6487 mg/kg bw/day in males and females, respectively.

In a further 2-year repeated dose toxicity study with Tween 80, 12 male and 20 female Wistar rats per group were treated daily with dietary concentrations of 5, 10 and 20%, corresponding to 3333, 6666 and 13333 mg/kg bw/day, respectively (Oser and Oser, 1956 and 1957). A similar constituted group of animals received the plain diet and served as controls. At 20% in the diet, higher mortality in females was observed compared to controls. However, at the same dietary concentration in males as well as at the two lower concentrations in both genders, no difference in the mortality of rats compared to controls was observed. In most males receiving 20% of the test substance in the diet, mild to moderate diarrhoea accompanied by mild or moderate to marked perianal irritation was observed, which was considered to be a laxative effect of the test substance at the highest dos level. Only very low incidences of laxation were seen in females of the low dose groups. Laxation was considered to be probably a cause of longer chain polyols resulting from hydrolysis of the test substance, which were also shown to induce laxation in rats in other studies.

No adverse effects on body weights and food consumption were observed in both genders at any dose level. No changes in organ weights (only liver and kidney were examined) and no substance-related findings at gross and histopathological examination were noted in treated animals of all dose groups. Furthermore, no adverse alterations in haematological parameters (haemoglobin, red blood cell count, white blood cell count, and differential blood cell count), clinical chemistry parameters (sugar, non-protein nitrogen, and plasma cholesterol) and urinary parameters (pH, presence of albumin, reducing sugars and microscopic appearance of sediment) were observed in treated animals.

Based on the results of this study, the NOAEL for male and female rats was considered to be 6666 mg/kg bw/day.

Conclusions for repeated dose oral toxicity

In a subacute repeated dose toxicity study in rats with Sorbitan monooleate, ethoxylated (Tween 81 [5EO]), feeding of diets containing 2000 and 4000 mg/kg bw/day (2 and 5% in the diet) did not result in any adverse effects on survival, body weights, organ weights of kidney and liver, white and red blood cell count and histopathology in livers, kidneys, intestines and bladders, in male rats.

More comprehensive data after repeated exposure are provided in subchronic and chronic toxicity studies with the structurally related substance Tween 80 (CAS 9005-65-6 polysorbat 80 [sorbitan monooleate ethoxylated (20EO)]).

In a 90-day feeding study in male and female F344/N rats similar to OECD 408, no adverse effects were identified after exposure to concentrations of up to 50000 ppm in the diet, corresponding 4132 and 6211 mg/kg bw/day in males and females, respectively.

A subsequent chronic feeding study in rats receiving dietary dose levels of up to 50000 ppm Tween 80 in the diet, corresponding to 2415 and 2745 mg/kg bw/day in males and females, respectively, did not result in any adverse effect on survival, body weights, food consumption and organ weights. However, a slight increase in the incidence of benign pheochromocytoma in adrenal medulla as well as an increased total incidence of pheochromocytoma in adrenal medulla of males was observed in males at the highest dose level. The slight increase in incidence of pheochromocytoma in the high dose group of males was judged to be of questionable significance, since the incidence of hyperplasia of the adrenal medulla, which is generally considered to be the precursor to pheochromocytoma, was only increased in the low dose group, but not the high dose group of males. Furthermore, no adverse effect on adrenals and any other organ were observed in females at any dose level. As there was an equivocal evidence of carcinogenic activity in adrenal medulla, the LOAEL for males was considered to be 50000 ppm, corresponding to 2415 mg/kg bw/day. The respective NOAEL for males was set at the lower dose level of 25000 ppm, which corresponded to 1174 mg/kg bw/day in males, which is above the current limit dose of 1000 mg/kg bw/day. Based on the lack of adverse findings, a NOAEL of ≥ 50000 ppm, corresponding to ≥ 2745 mg/kg bw/day was derived for females.

A further chronic toxicity study with Tween 80 in mice receiving the same dietary concentrations as described in the chronic study in rats resulted in a statistically significant increase in the incidence of inflammation and squamous hyperplasia (higher severity) of the forestomach of both genders as well as a statistically significant increase in the incidence of ulcers in females at 50000 ppm. Based on these results, a NOAEL of 5003 and 6487 mg/kg bw/day was derived for males and females, respectively.

Additional information on chronic toxicity of Tween 80 via the oral route is provided in a further feeding study in rats, resulting in no adverse effects up to a dietary dose level of 6666 mg/kg bw/day in males and females, respectively. Only a low incidence of diarrhoea was observed in a female which was considered to be a treatment-related, but not adverse effect in these animals. At the highest dose level of 13333 mg/kg bw/day, most males showed mild to moderate diarrhoea accompanied by mild/moderate to marked perianal irritation. At the same dose level, a higher mortality in females was observed compared to treated males of the same dose group and the controls. Based on the results on this study, the LOAEL for rats was set at 13333 mg/kg bw/day.

Overall, the available data on repeated dose toxicity of the test substance and the structurally related substance Tween 80 (CAS 9005-65-6 polysorbat 80 [sorbitan monooleate ethoxylated (20EO)]) consistently resulted in NOAELs greater than 1000 mg/kg bw/day. Therefore, the potential of the substance to induce adverse toxic effects after repeated oral exposure under normal conditions of use is considered to be low.

There is no data available on repeated dose toxicity by the inhalation and dermal routes.

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 repeated dose toxicity via oral route - systemic effects endpoint:
Hazard assessment is based on the weight of evidence from all available studies. Therefore, no endpoint selection was made.

Justification for classification or non-classification

Based on substance-specific studies and read-across from a structurally similar substance, the available data on the repeated dose toxicity via the oral route 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.

There is no data available on repeated dose toxicity by the inhalation and dermal routes.