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

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Quality of whole database:

The available information comprises an adequate and reliable study (Klimisch score 2 due to read-across) 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). The selected study is thus sufficient to fulfil the standard information requirements set out in Annex VIII - IX, 8.7, in accordance with Annex XI,1.5, of Regulation (EC) No 1907/2006.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Toxicity to reproduction

Justification for grouping of substances and read-across

There are no data available on toxicity to reproduction (fertility) 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, 8.7.3., 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, 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 thepolyethoxylated 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, thepolyethoxylated 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

Toxicity to reproduction - Fertility (three-generation study)

CAS9005-65-6 (Tween 80 (CAS 9005-65-6 polysorbat 80 [sorbitan monooleate ethoxylated (20EO)]) )

In a three-generation reproduction toxicity study, Tween 80 was administered 12 weeks prior to mating and then continuously over a 2-year feeding period to 12 male and 20 female Wistar rats of the F0 generation at dietary concentrations of 5, 10 and 20%, corresponding to 3333, 6666 and 13333 mg/kg bw/day based on the assumption of an average food consumption of 20 g/animal and an average body weight of 300 g (Oser and Oser, 1956 and 1957). Ten animals per sex were selected from the second litter of each following generation (F1, F2 and F3) and treated continuously over the remaining 2-year feeding period at the same dose levels like the initial F0 generation. In all generations, a corresponding control group was treated with the plain diet over the same feeding period. The selection of parental animals from each generation was performed when animals were 21 days of age.

During the study period, a higher mortality in females was observed at the 20% dietary concentration in the F0 generation when compared to controls. At the same dietary concentration in males as well as at the two lower concentrations in both genders, no difference in the mortality of F0 rats compared to controls was observed. The median survival time (MST) of the F0 generation, expressed as the age in weeks when one-half of a group had died, revealed that the MST of the treated animals of all dose groups values fell within the MST of the controls. Therefore, the MST was not affected by either the dietary level of any of the test substance, nor when compared with those of the controls.

In most F0 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. 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.

In all generations, no significant effects on body weights and food consumption were observed. At necropsy, no changes in organ weights (only liver and kidney were examined) and no substance-related findings at gross and histopathological examination were noted in all generations.

At the highest dose group (20% of the test substance in the diet), reduced fertility was observed in the F0 generation. This finding was also associated with irritation and hypersensitivity around the anus due to the laxative effect of the test substance at this concentration, probably resulting in lack of interest in copulation.

In the F0 generation, at all dose levels, no effect on gestation was observed in the treated animals compared to controls. However, there was a tendency toward diminished productivity (in terms of numbers of litters) in groups receiving the test substance at the 20% level, which could be due to laxative effects of the test substance at this dose level resulting in lack of interest in copulation.

Comparison of the reproduction data showed that the responses in the three successive generations were quite similar. The proportion of matings resulting in pregnancy (fertility index) tended to be lower at the 20% dietary level of the test substance. No adverse or cumulative effect on mating instinct, fertility or gestation occurred in any of the groups of rats receiving the same test or control rations through three successive generations. Reproductive performance in general appeared to be inferior in the third generation (F2) rats compared to their progenitors.

The reproductive performance of the F3 generation was not investigated because the rats in this generation were sacrificed either at weaning or at the end of the 12-week growth period.

In all generations, the size of the litters was substantially reduced at weaning in all groups (treated and controls), which occurred principally during the few days immediately after birth. At the 20% dietary level, a significant reduction in survival of newborns was observed in all generations. The trend toward higher mortality during the 4 days post-partum was not as marked in the F1 and F2 as in the F0 generation.

At the 20% dietary level, slightly lower pub weights at weaning in all generations was observed compared to controls which is normally associated with a possible impairment of lactation. However, neglect of the litters by maternal animals was rather associated with this effect than lactation failure per se, since no significant effects on the lactation indices were observed at this dose level compared to controls. The neglect of litters by maternal animals was considered to be a result of the laxative effect of the test substance at 20% in the diet, resulting in posterior ventral irritation that may have caused adverse influence on the interest of dams in caring for their offspring.

Based on the results of the study, the NOAEL for effects on systemic toxicity, fertility, and developmental toxicity was considered to be 10% in the diet, which corresponds to 6666 mg/kg bw/day. As the test substance was associated with laxative effects in males, a higher mortality rate in F0 females, slight reduction in fertility indices, and significant reduction in survival of new born at 4 days post-partum, the LOAEL for systemic effects, fertility and developmental toxicity was considered to be 13333 mg/kg bw/day (20% in the diet).

 

Conclusions for toxicity to reproduction (fertility)

One three-generation study over a period of 2 years in rats is available for Tween 80 (CAS 9005-65-6 polysorbat 80 [sorbitan monooleate ethoxylated (20EO)]), which resulted in a LOAEL of 13333 mg/kg bw/day for systemic effects, fertility and developmental toxicity based on laxative effects in males, a higher mortality rate in F0 females, slight reduction in fertility indices, and significant reduction in survival of new born at 4 days post-partum. Based on no adverse effects at the lower dietary dose levels of 3333 and 6666 mg/kg bw/day, the NOAEL was set at 6666 mg/kg bw/day, which is considerably above the currently applied limit dose of 1000 mg/kg bw/day.

 

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

 

 

 

Short description of key information:
NOAEL oral (rat, fertility): 6666 mg/kg bw/day (CAS 9005-65-6, polysorbat 80 [sorbitan monooleate ethoxylated (20EO)]; Tween 80 )
LOAEL oral (rat, fertility): 13 333 mg/kg bw/day (CAS 9005-65-6, polysorbat 80 [sorbitan monooleate ethoxylated (20EO)]; Tween 80 )

Justification for selection of Effect on fertility via oral route:
Hazard assessment is conducted by means of read-across from a structural analogue. The selected study is a reliable study based on the identified similarities in structure and intrinsic properties between source and target substance and overall quality assessment (refer to the endpoint discussion for further details). As the effects observed on fertility, systemic and developmental toxicity occur at a concentration of 13 333 mg/kg bw/day which is considerably above the currently applied limit dose, no hazard potential on fertility is expected under normal conditions of use.

Effects on developmental toxicity

Description of key information

NOAEL oral (rat, developmental): ≥ 5000 mg/kg bw/day
NOAEL oral (mouse, developmental): ≥ 2500 mg/kg bw/day

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Quality of whole database:

The available information comprises 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). The information from these independent sources is consistent and provides sufficient weight of evidence for hazard assessment leading to an endpoint conclusion in accordance with Annex XI, 1.2, of Regulation (EC) No 1907/2006. Therefore, the available information as a whole is sufficient to fulfil the standard information requirements set out in Annex VIII-IX 8.7, of Regulation (EC) No 1907/2006.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Developmental toxicity

Justification for grouping of substances and read-across

There are no data available on developmental 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, 8.7.2., 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 1 – 5 or 20, 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 sorbitanmoiety, 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

Prenatal developmental toxicity

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

A developmental toxicity study similar to OECD 414 was performed with Tween 80 in Sprague-Dawley rats (Price et al., 1992). Twenty-five time-mated females per group received the test substance in aqueous solution (deionised/distilled water) via oral gavage from gestation day (GD) 6 to 15 at dose levels of 500 and 5000 mg/kg bw/day, respectively. A similar constituted group of time-mated females was treated with the vehicle only and served as controls.

At both dose levels, no differences in the number of corpora lutea per dam, the number of implantation sites per dam or the percent pre-implantation loss per litter were observed compared to controls. Furthermore, no adverse effects were noted on the growth and viability of the foetuses.

No adverse effects were noted on growth, viabilty or morphological development of fetuses. The overall incidence of malformations did not differ significantly among the treatment groups. Moreover, only a slight increase in the incidence of fetuses showing external, visceral or anatomical variations was determined in test animals after intrauterine exposure to 500 or 5000 mg/kg bw/day: However, except the skeletal anomalies, no dose-relationship was observed and hence, visceral and external effects are considered as incidental and not treatment-related. In the low dose group, one foetus (1/280 or 0.4%) exhibited any skeletal malformations visible as bipartite vertebra in the thoracic region (i.e. bipartite cartilage and bipartite ossification centres) which is a common finding in this species and strain. At 5000 mg/kg bw/day, 4 foetuses (4/341 or 1.2%) exhibited skeletal malformations: 3 foetuses exhibited only bipartite vertebra in the thoracic region (i.e. two with bipartite cartilage and bipartite ossification centres and one with bipartite cartilage and dumbbell ossification centre) and one foetus exhibited multiple skeletal malformations (agenesis of the lumbar, sacral and caudal vertebra; fused thoracic vertebra; fused ribs, ribs unattached to the sternum) accompanied by multiple external malformations (craniorhachischisis, short tail and umbilical hernia). However, as the fetal incidence of abnormal skeletal findings lay within the range of spontaneous skeletal anomalies determined on gestational day 20 (MARTA, 1993), the determined skeletal abnormalities are not interpreted as adverse but rather spontaneous.

No treatment-related mortalities and clinical signs of toxicity were observed in the dams in any dose group. Average maternal body weight did not differ among treatment groups, nor was there a treatment related change in maternal weight gain during treatment or gestation. Maternal food intake was comparable across all groups during the study period, but was decreased by -14% during the first 3 days of treatment at 5000 mg/kg bw/day relative to the vehicle control group. Maternal relative water intake was comparable among treatment groups throughout gestation.

At sacrifice, no treatment-related effects on the organ weights of gravid uterine (absolute), right kidney (absolute or relative) and heart (absolute or relative) were observed in dams. In contrast, relative maternal liver weight was statistically significantly elevated in both treatment groups, whereas absolute liver weight was only significantly increased at 500 mg/kg bw/day. However, due to the low increase in relative organ weight and the absence of any data on histopathological changes in liver, this effect was considered to be non-adverse.

As no adverse effects on dams were observed during the study, the NOAEL for maternal toxicity was determined to be ≥ 5000 mg/kg bw/day. Moreover, no adverse effects were noted on growth, viabilty or morphological development of foetuses and hence, a NOAEL of be ≥ 5000 mg/kg bw/day was defined for developmental toxicity.

A further prenatal developmental toxicity study with Tween 80 was performed in 30 time-pregnant female CD-1 mice, which received the test substance in vehicle at a dose level of 2500 mg/kg bw/day during gestation Days 8-12 (Kavlock et al., 1987). A further group of 40 pregnant females was treated with the vehicle only and served as controls. Up to gestation Day 20, no effects on maternal survival and body weight gain were observed. At post-mortem examination of the dams, no resorptions were seen in treated and control groups. Furthermore, no effects on early postnatal growth and viability of the pubs were observed. Based on the results of this study, the NOAEL for maternal and developmental toxicity for mice was considered to be ≥ 2500 mg/kg bw/day.

Conclusions for toxicity to reproduction (development)

A pre-natal developmental toxicity study with Tween 80 (CAS 9005-65-6 polysorbat 80 [sorbitan monooleate ethoxylated (20EO)]) is available, in which the substance was administered to female rats in the diet at dose levels of 500 and 5000 mg/kg bw/day from gestation day (GD) 6 to 15. As no adverse effects were observed on dams and foetuses, Tween 80 (CAS 9005-65-6 polysorbat 80 [sorbitan monooleate ethoxylated (20EO)]) is not considered to negatively affect intrauterine development. A further pre-natal developmental toxicity study in mice is available, which did not show any effects on the survival and growth of newborns and no maternal toxicity up to 2500 mg/kg bw/day.

 

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 Effect on developmental toxicity: via oral route:
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 the read-across from a structurally similar substance, the available data on toxicity to reproduction 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.