Squalene-rich fraction obtained from vegetable oil deodorizer distillate by transesterification, crystallisation and vacuum distillation

Repeated dose toxicity: dermal

Administrative data

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Registration/ Notification status for the use:

use registered according to REACH Article 10; total tonnage manufactured/imported >=10tonnes/year per registrant

Use name:

Use as intermediate in the manufacture of other substances (squalane)

Name of activity / technique:

Use as intermediate in the manufacture of other substances (squalane)

Environmental release category (ERC):

ERC6a: Use of intermediate

Name of activity / technique:

Closed process without likelihood of exposure

Process category (PROC):

PROC 1: Chemical production or refinery in closed process without likelihood of exposure or processes with equivalent containment conditions

Name of activity / technique:

Closed continuous process with occasional controlled exposure

Process category (PROC):

PROC 2: Chemical production or refinery in closed continuous process with occasional controlled exposure or processes with equivalent containment conditions

Name of activity / technique:

Closed batch process with occasional controlled exposure

Process category (PROC):

PROC 3: Manufacture or formulation in the chemical industry in closed batch processes with occasional controlled exposure or processes with equivalent containment conditions

Name of activity / technique:

Transfer of the substance at dedicated facilities

Process category (PROC):

PROC 8b: Transfer of substance or mixture (charging and discharging) at dedicated facilities

Name of activity / technique:

Transfer of substance or mixture into small containers

Process category (PROC):

PROC 9: Transfer of substance or mixture into small containers (dedicated filling line, including weighing)

Name of activity / technique:

Laboratory analysis of samples

Process category (PROC):

PROC 15: Use as laboratory reagent

Substance supplied to that use in form of:

as such

Subsequent service life relevant for this use:

no

Related assessment:

use assessed in an own CSR

Linked contributing activity:

01710c11-e21d-4639-bd3b-cb21416ab2c9

Biological Sewage Treatment Plant (STP):

no

Particular considerations on the waste treatment operations:

no

Receiving surface water flow rate (m³/day):

18 000

Other conditions of use impacting on environmental exposure:

Discharge rate of effluent: >= 2E3 m3/day

Release from the site to::

surface water

Release estimation method:

estimated release factor

Remarks:

Industry-specific

Release factor from the site after on-site risk management (%):

0

Release from the site to::

ambient air

Release estimation method:

estimated release factor

Remarks:

Industry-specific

Release factor from the site after on-site risk management (%):

0

Release from the site to::

non-agricultural soil

Release estimation method:

estimated release factor

Remarks:

Industry-specific

Release factor from the site after on-site risk management (%):

0

Release factor to external waste (%):

0

Exposed compartment:

freshwater

Clocal:

0 mg/L

Predicted exposure concentration:

0 mg/L

Exposure estimation method:

exposure estimation tool

Exposure estimation tool name:

EUSES

Version of exposure estimation tool:

2.1.2

Exposed compartment:

sediment (freshwater)

Predicted exposure concentration:

0 mg/kg dw

Exposure estimation method:

exposure estimation tool

Exposure estimation tool name:

EUSES

Version of exposure estimation tool:

2.1.2

Exposed compartment:

marine water

Clocal:

0 mg/L

Predicted exposure concentration:

0 mg/L

Exposure estimation method:

exposure estimation tool

Exposure estimation tool name:

EUSES

Version of exposure estimation tool:

2.1.2

Exposed compartment:

sediment (marine water)

Predicted exposure concentration:

0 mg/kg dw

Exposure estimation method:

exposure estimation tool

Exposure estimation tool name:

EUSES

Version of exposure estimation tool:

2.1.2

Exposed compartment:

sewage treatment plant

Predicted exposure concentration:

0 mg/L

Exposure estimation method:

exposure estimation tool

Exposure estimation tool name:

EUSES

Version of exposure estimation tool:

2.1.2

Exposed compartment:

air

Clocal:

0 mg/m³

Predicted exposure concentration:

0 mg/m³

Exposure estimation method:

exposure estimation tool

Exposure estimation tool name:

EUSES

Version of exposure estimation tool:

2.1.2

Exposed compartment:

agricultural soil

Clocal:

0 mg/kg dw

Predicted exposure concentration:

0 mg/kg dw

Exposure estimation method:

exposure estimation tool

Exposure estimation tool name:

EUSES

Version of exposure estimation tool:

2.1.2

Exposed compartment:

predators' prey (freshwater)

Predicted exposure concentration:

0 mg/kg ww

Exposure estimation method:

exposure estimation tool

Exposure estimation tool name:

EUSES

Version of exposure estimation tool:

2.1.2

Exposed compartment:

predators' prey (marine water)

Predicted exposure concentration:

0 mg/kg ww

Exposure estimation method:

exposure estimation tool

Exposure estimation tool name:

EUSES

Version of exposure estimation tool:

2.1.2

Exposed compartment:

top predators' prey (marine water)

Predicted exposure concentration:

0 mg/kg ww

Exposure estimation method:

exposure estimation tool

Exposure estimation tool name:

EUSES

Version of exposure estimation tool:

2.1.2

Exposed compartment:

predators' prey (terrestrial)

Predicted exposure concentration:

0 mg/kg ww

Exposure estimation method:

exposure estimation tool

Exposure estimation tool name:

EUSES

Version of exposure estimation tool:

2.1.2

Daily intake via food consumption:

0 mg/kg bw/day

Exposure estimation method:

exposure estimation tool

Exposure estimation tool name:

EUSES

Version of exposure estimation tool:

2.1.2

Linked contributing activity:

c55aa072-b928-4f08-956e-be488769fc85

Percentage (w/w) of substance in mixture/article:

100

Physical form of the used product:

liquid, including paste/slurry/suspension

Duration of activity (hour/day):

8

Technical and organisational conditions and measures:

General ventilation: Basic general ventilation (1-3 air changes per hour)

Effectiveness (%):

0

Route affected:

inhalation

Technical and organisational conditions and measures:

Occupational Health and Safety Management System: Advanced

Technical and organisational conditions and measures:

Local exhaust ventilation: No

Effectiveness (%):

0

Route affected:

inhalation

Effectiveness (%):

0

Route affected:

dermal

Personal protection equipment:

Respiratory protection: No

Effectiveness (%):

0

Route affected:

inhalation

Personal protection equipment:

Dermal protection: No

Effectiveness (%):

0

Route affected:

dermal

Place of use:

indoor

Operating temperature (°C):

40

Type of exposure:

inhalation (external) long-term exposure

Exposure estimate:

0.208 mg/m³

Exposure estimation method:

exposure estimation tool

Exposure estimation tool name:

ECETOC TRA workers

Version of exposure estimation tool:

3.0

Type of exposure:

inhalation (external) short-term exposure

Exposure estimate:

0.208 mg/m³

Exposure estimation method:

exposure estimation tool

Exposure estimation tool name:

ECETOC TRA workers

Version of exposure estimation tool:

3.0

Type of exposure:

dermal (external) long-term exposure

Exposure estimate:

0.034 mg/kg bw/day

Exposure estimation method:

exposure estimation tool

Exposure estimation tool name:

ECETOC TRA workers

Version of exposure estimation tool:

3.0

Linked contributing activity:

62f9ad30-937e-46a0-939f-d99b683975da

Percentage (w/w) of substance in mixture/article:

100

Physical form of the used product:

liquid, including paste/slurry/suspension

Duration of activity (hour/day):

8

Technical and organisational conditions and measures:

General ventilation: Basic general ventilation (1-3 air changes per hour)

Effectiveness (%):

0

Route affected:

inhalation

Technical and organisational conditions and measures:

Occupational Health and Safety Management System: Advanced

Technical and organisational conditions and measures:

Local exhaust ventilation: No

Effectiveness (%):

0

Route affected:

inhalation

Effectiveness (%):

0

Route affected:

dermal

Personal protection equipment:

Respiratory protection: No

Effectiveness (%):

0

Route affected:

inhalation

Personal protection equipment:

Dermal protection: No

Effectiveness (%):

0

Route affected:

dermal

Place of use:

indoor

Operating temperature (°C):

40

Type of exposure:

inhalation (external) long-term exposure

Exposure estimate:

2.083 mg/m³

Exposure estimation method:

exposure estimation tool

Exposure estimation tool name:

ECETOC TRA workers

Version of exposure estimation tool:

3.0

Type of exposure:

inhalation (external) short-term exposure

Exposure estimate:

2.083 mg/m³

Exposure estimation method:

exposure estimation tool

Exposure estimation tool name:

ECETOC TRA workers

Version of exposure estimation tool:

3.0

Type of exposure:

dermal (external) long-term exposure

Exposure estimate:

1.37 mg/kg bw/day

Exposure estimation method:

exposure estimation tool

Exposure estimation tool name:

ECETOC TRA workers

Version of exposure estimation tool:

3.0

Linked contributing activity:

49b22a54-be7e-4a72-b57e-5d7425954ccf

Percentage (w/w) of substance in mixture/article:

100

Physical form of the used product:

liquid, including paste/slurry/suspension

Duration of activity (hour/day):

8

Technical and organisational conditions and measures:

General ventilation: Basic general ventilation (1-3 air changes per hour)

Effectiveness (%):

0

Route affected:

inhalation

Technical and organisational conditions and measures:

Occupational Health and Safety Management System: Advanced

Technical and organisational conditions and measures:

Local exhaust ventilation: No

Effectiveness (%):

0

Route affected:

inhalation

Effectiveness (%):

0

Route affected:

dermal

Personal protection equipment:

Respiratory protection: No

Effectiveness (%):

0

Route affected:

inhalation

Personal protection equipment:

Dermal protection: No

Effectiveness (%):

0

Route affected:

dermal

Place of use:

indoor

Operating temperature (°C):

40

Type of exposure:

inhalation (external) long-term exposure

Exposure estimate:

2.083 mg/m³

Exposure estimation method:

exposure estimation tool

Exposure estimation tool name:

ECETOC TRA workers

Version of exposure estimation tool:

3.0

Type of exposure:

inhalation (external) short-term exposure

Exposure estimate:

2.083 mg/m³

Exposure estimation method:

exposure estimation tool

Exposure estimation tool name:

ECETOC TRA workers

Version of exposure estimation tool:

3.0

Type of exposure:

dermal (external) long-term exposure

Exposure estimate:

0.69 mg/kg bw/day

Exposure estimation method:

exposure estimation tool

Exposure estimation tool name:

ECETOC TRA workers

Version of exposure estimation tool:

3.0

Linked contributing activity:

6e475610-dca6-41de-85d4-6254f819e10a

Percentage (w/w) of substance in mixture/article:

100

Physical form of the used product:

liquid, including paste/slurry/suspension

Duration of activity (hour/day):

8

Technical and organisational conditions and measures:

General ventilation: Basic general ventilation (1-3 air changes per hour)

Effectiveness (%):

0

Route affected:

inhalation

Technical and organisational conditions and measures:

Occupational Health and Safety Management System: Advanced

Technical and organisational conditions and measures:

Local exhaust ventilation: No

Effectiveness (%):

0

Route affected:

inhalation

Effectiveness (%):

0

Route affected:

dermal

Personal protection equipment:

Respiratory protection: No

Effectiveness (%):

0

Route affected:

inhalation

Personal protection equipment:

Dermal protection: Yes (Chemically resistant gloves conforming to EN374) and (other) appropriate dermal protection

Effectiveness (%):

80

Route affected:

dermal

Place of use:

indoor

Operating temperature (°C):

40

Type of exposure:

inhalation (external) long-term exposure

Exposure estimate:

2.083 mg/m³

Exposure estimation method:

exposure estimation tool

Exposure estimation tool name:

ECETOC TRA workers

Version of exposure estimation tool:

3.0

Type of exposure:

inhalation (external) short-term exposure

Exposure estimate:

2.083 mg/m³

Exposure estimation method:

exposure estimation tool

Exposure estimation tool name:

ECETOC TRA workers

Version of exposure estimation tool:

3.0

Type of exposure:

dermal (external) long-term exposure

Exposure estimate:

2.742 mg/kg bw/day

Exposure estimation method:

exposure estimation tool

Exposure estimation tool name:

ECETOC TRA workers

Version of exposure estimation tool:

3.0

Linked contributing activity:

908f15ff-2afa-4c8b-acb9-dc02866e4583

Percentage (w/w) of substance in mixture/article:

100

Physical form of the used product:

liquid, including paste/slurry/suspension

Duration of activity (hour/day):

8

Technical and organisational conditions and measures:

General ventilation: Basic general ventilation (1-3 air changes per hour)

Effectiveness (%):

0

Route affected:

inhalation

Technical and organisational conditions and measures:

Occupational Health and Safety Management System: Advanced

Technical and organisational conditions and measures:

Local exhaust ventilation: No

Effectiveness (%):

0

Route affected:

inhalation

Effectiveness (%):

0

Route affected:

dermal

Personal protection equipment:

Respiratory protection: No

Effectiveness (%):

0

Route affected:

inhalation

Personal protection equipment:

Dermal protection: Yes (Chemically resistant gloves conforming to EN374) and (other) appropriate dermal protection

Effectiveness (%):

80

Route affected:

dermal

Place of use:

indoor

Operating temperature (°C):

40

Type of exposure:

inhalation (external) long-term exposure

Exposure estimate:

2.083 mg/m³

Exposure estimation method:

exposure estimation tool

Exposure estimation tool name:

ECETOC TRA workers

Version of exposure estimation tool:

3.0

Type of exposure:

inhalation (external) short-term exposure

Exposure estimate:

2.083 mg/m³

Exposure estimation method:

exposure estimation tool

Exposure estimation tool name:

ECETOC TRA workers

Version of exposure estimation tool:

3.0

Type of exposure:

dermal (external) long-term exposure

Exposure estimate:

1.372 mg/kg bw/day

Exposure estimation method:

exposure estimation tool

Exposure estimation tool name:

ECETOC TRA workers

Version of exposure estimation tool:

3.0

Linked contributing activity:

adbb84a9-50b8-410f-bd42-b1f3f52e980e

Percentage (w/w) of substance in mixture/article:

100

Physical form of the used product:

liquid, including paste/slurry/suspension

Duration of activity (hour/day):

8

Technical and organisational conditions and measures:

General ventilation: Basic general ventilation (1-3 air changes per hour)

Effectiveness (%):

0

Route affected:

inhalation

Technical and organisational conditions and measures:

Occupational Health and Safety Management System: Advanced

Technical and organisational conditions and measures:

Local exhaust ventilation: No

Effectiveness (%):

0

Route affected:

inhalation

Effectiveness (%):

0

Route affected:

dermal

Personal protection equipment:

Respiratory protection: No

Effectiveness (%):

0

Route affected:

inhalation

Personal protection equipment:

Dermal protection: Yes (Chemically resistant gloves conforming to EN374) and (other) appropriate dermal protection

Effectiveness (%):

80

Route affected:

dermal

Place of use:

indoor

Operating temperature (°C):

40

Type of exposure:

inhalation (external) long-term exposure

Exposure estimate:

2.083 mg/m³

Exposure estimation method:

exposure estimation tool

Exposure estimation tool name:

ECETOC TRA workers

Version of exposure estimation tool:

3.0

Type of exposure:

inhalation (external) short-term exposure

Exposure estimate:

2.083 mg/m³

Exposure estimation method:

exposure estimation tool

Exposure estimation tool name:

ECETOC TRA workers

Version of exposure estimation tool:

3.0

Type of exposure:

dermal (external) long-term exposure

Exposure estimate:

0.068 mg/kg bw/day

Exposure estimation method:

exposure estimation tool

Exposure estimation tool name:

ECETOC TRA workers

Version of exposure estimation tool:

3.0

Cross-reference 2

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Based on the different toxicokinetic studies on the constituents, the test substance ‘squalene-rich fraction obtained from vegetable oil deodorizer distillate by transesterification, crystallisation and vacuum distillation’ is considered to be moderately to poorly absorbed depending upon the composition. The extent of absorption of glycerides, fatty acids and fatty acids methyl esters in the gastro-intestinal system varies depending on the chain length and their degree of saturation. Generally, short-chain fatty acids are better absorbed than the long chain counterparts. Their skin permeability increases with the lipophilic nature of a compound. The tocopherols from the unsaponifiable matters were considered to be well absorbed through oral (21-86%) as well as dermal routes. However, tissue tocopherol contents tend to be related exponentially to vitamin E intake and showed no deposition or saturation thresholds. As a result, tissues vary considerably in tocopherol contents in a manner that is not related to their lipid content. Further, the sterols, sterol esters, hydrocarbons and squalene were generally poorly absorbed from the gastro-intestinal tract and slowly absorbed through the skin.

Overall, based on the toxicokinetic studies on the constituents and considering the exposed amount under the normal use conditions, the test substance ‘squalene-rich fraction obtained from vegetable oil deodorizer distillate by transesterification, crystallisation and vacuum distillation’ is not expected to bioaccumulate. Further, in the absence of quantitative absorption values for all the constituent types and as a conservative approach, 100% absorption rates have been considered for the hazard assessment. Additionally, for route-to-route extrapolations, default absorption factors have been used as per the ECHA guidance.

Bioaccumulation potential:

low bioaccumulation potential

Absorption rate - oral (%):

100

Absorption rate - dermal (%):

100

Absorption rate - inhalation (%):

100

In the absence of direct toxicokinetics data with the test substance, ‘squalene-rich fraction obtained from vegetable oil deodorizer distillate by transesterification, crystallisation and vacuum distillation’ the endpoint has been assessed based on studies for substances representative of the main constituents, which can be categorised as glycerides, fatty acids or fatty acid methyl esters and unsaponifiable matter (including tocopherols, sterols, squalene and hydrocarbons). As the individual constituents are data rich because of their nutritional and cosmetic uses, for practical reasons, only a limited number of studies are reported below:

Glycerides:

When taken up orally, glycerides are split in the intestinal lumen into glycerol and fatty acids with the help of lipases and bile secretions, then move into enterocytes. The triglycerides are rebuilt in the enterocytes from their fragments and packaged together with cholesterol and proteins to form chylomicrons. These are excreted from the cells, collected by the lymph system and transported to the large vessels near the heart before entering the blood. Eventually, the triglycerides bind to the membranes of hepatocytes, adipocytes or muscle fibers, where they are either stored or oxidized for energy. When the body requires fatty acids as a source of energy, the hormone glucagon signals the breakdown of the triglycerides by hormone-sensitive lipases to release free fatty acids. The fatty acids are then broken down by stepwise elimination of C2-units in the mitochondrial β-oxidation. Alternate oxidation pathways can be found in the liver (ω-oxidation) and the brain (α-oxidation) (HERA, 2002).The C2 -units are esterified to acetyl-coenzyme A which directly enters the citric acid cycle where it is converted to carbon dioxide and energy (MacDonald, 1973; Robinson, 1973; Chen and Farese, 2002). Not all fatty acids present as triglycerides are used for energy production: after metabolism in the liver, redistribution to phospholipids and sterol esters may, for example, also occur (Mead and Fillerup, 1957; McArthuret al., 1999).

Glycerides with alkyl chain lengths between C8 to18, including C18-unsatd. are generally poorly water soluble have an estimated log Pow > 6 and molecular weights > 500. As such, uptake into the stratum corneum and further transfer into the epidermis are likely to be low (REACH guidance document R7.C (May 2008)).

Fatty acids:

Upon ingestion, fatty acids are directly taken up into the cells lining the intestines (enterocytes), then transported mainly in the form of triglycerides via the lymph to various tissues (see below). The fatty acids may then be stored in the form of triglycerides as a source of energy or redistributed to phospholipids and sterol esters (conversion mainly in the liver) (Mead and Fillerup, 1957; McArthur et al., 1999).

The extent of absorption in the gastro-intestinal system varies depending on the chain length of the fatty acids and their degree of saturation. Generally, short-chain fatty acids are better absorbed than the long chain counterparts. Also, absorption decreases with increasing saturation (MacDonald, 1973; Robinson, 1973; Chen and Farese, 2002). In an overview by the Cosmetic Ingredient Review Panel (CIR, 1987), stearic acid (C18) was cited as being the most poorly absorbed of the common fatty acids.

Only limited information could be located on dermal penetration of fatty acids. In dermal application studies in the rat (Butcher, 1951), linoleic acid was shown to penetrate the epithelium rapidly and reach the vascular system. Oleic acid was also reported to penetrate the epithelium of rats, possibly via hair follicles, but only minute amounts were seen in the blood vessels. Ricinoleic acid, on the other hand, was retained mainly in the outer strata of the epidermis. Other authors have noted that skin permeability increases with the lipophilic nature of a compound (Scheuplein, 1965; CIR, 1987). Dermal uptake of fatty acids has also been studied with fatty acid soaps. The C10and C12 soaps show the greatest skin penetration of human epidermis. Also, percutaneous absorption of sodium laurate is greater than that of most other anionic surfactants (HERA, 2002).

Fatty acid methyl esters:

Oral administration of radiolabeled ethyl oleate and triolein in Sprague–Dawley rats after a single, peroral dose of 1.7 or 3.4 g/kg bw were found to be well absorbed with approximately similar extent of absorption (ca. 70–90% and 90-100% absorption respectively). At sacrifice (72 h post-dose), tissue distribution of test substance-derived radioactivity and TG-derived radioactivity was similar, with maximum radioactivity found in mesenteric fat. Both test substances were rapidly and extensively excreted as CO2 with no remarkable differences between their excretion profiles. Approximately 40–70% of the administered dose for both groups were excreted as CO2 within the first 12 h (consistent with b-oxidation of fatty acids). Fecal elimination of test substance appeared to be dose-dependent, ranging from 7-20% for ethyl oleate and 2-4% for triolein (Bookstaff, 2003).

Fatty acids methyl esters are expected to be hydrolysed to the corresponding alcohol (methanol) and fatty acid by esterases (Fukami and Yokoi, 2012), even though it was shown in-vitro that the hydrolysis rate of methyl oleate was lower when compared with the hydrolysis rate of the triglyceride Glycerol trioleate (Mattson and Volpenhein, 1972). The resulting fatty acids will be metabolized via beta-oxidation, while methanol will be slowly oxidized in the liver by the enzyme alcohol dehydrogenase (ADH) to formaldehyde, which itself is oxidized very rapidly by the enzyme aldehyde dehydrogenase (ALDH) to formic acid. Finally, formic acid is slowly metabolised to CO2 and H2O (ICPS, 2002).

Little information could be found on absorption of fatty acid methyl esters via the dermal route. However, given the log Kow predictions for the representative substances, which is estimated to be >3, fatty acid methyl ester constituents are not expected to be taken up through skin to a significant degree (as per Table R 7.12 -3, REACH Endpoint specific guidance R.7c of May 2008).

Unsaponifiable matter:

Tocopherols:

The toxicokinetics of tocopherols has been extensively studied in humans, in particular due to the Vitamin E activity. α-tocopherol is the most active of all homologues, followed by β-, γ-, and δ-tocopherol. Only certain isomers are retained in human plasma, i.e. the RRR-α-tocopherol and the 2R-stereoisomers, RSR, RRS- and RSS-α-tocopherol (Traber, 1999).

Upon oral administration, α-tocopherol is absorbed unchanged from the small intestine by passive diffusion. Tocotrienol esters are first hydrolysed by pancreatic esterase (Bjørneboe et al., 1990). Absorption occurs mostly in the upper and middle thirds of the small intestine (Tomassi and Silano, 1986; Fiume, 2002) and the absorbed substance enters the lymphatic circulation (Devron, 1999). The uptake efficiency of tocopherol and its esters is generally considered to be variable. In rats given a single bolus of α-tocopherol intraduodenally, absorption was reported to be approximately 40% (Bjørneboe et al., 1990), whereas when α-tocopherol acetate was given as slow continuous infusion into the duodenum, absorption was 65% (Traber et al., 1986). In another study, the appearance of α-tocopherol in the lymph was negligible in the 2-4 h following intraduodenal dosing, reaching its peak 4-15 h after feeding (Bjørneboe et al., 1986). Intestinal absorption via the lymphatic system was 15.4%. In human studies over 24 h, absorption of α-tocopherol and its acetate ester was in the range of 21-86%. However, determination under experimental conditions may not reflect dietary reality.

α-tocopherol is rapidly transferred in plasma from chylomicrons to plasma lipoproteins, to which it binds non-specifically. The vitamin is taken up by the liver and released in low density lipoprotein (Traber et al., 1988; Combs, 1992). Most absorbed tocopherols are transported unchanged to the tissues. In non-adipose cells, vitamin E is localised almost exclusively in the membranes. Kinetic studies indicate that such tissues have two pools of the vitamin: a ’labile’, rapidly turning over pool, and a fixed, slowly turning over pool. The labile pools predominate in such tissues as plasma and liver, as the tocopherol contents of those tissues are depleted rapidly under conditions of vitamin E deprivation. In contract, the adipose vitamin E resides predominately in the bulk lipid phase, which appears to be a fixed pool of vitamin, thus, it is only slowly metabolised from this tissue (Bjørneboe et al., 1990, Combs, 1992; Basu and Dickerson, 1996). Tissue tocopherol contents tend to be related exponentially to vitamin E intake and show no deposition or saturation thresholds. As a result, tissues vary considerably in tocopherol contents in a manner that is not related to their lipid content (Machlin, 1984). Tocopherols are generally well absorbed through human skin (Fiume, 2002).

Sterols and sterol esters:

Observations in animals and humans have shown that plant sterols and sterol esters are generally poorly absorbed when taken up orally, with the highest absorption occurring for campesterol. Consumption of sterols nevertheless leads to a small but dose-related increase in plasma concentrations in short-term studies. In repeated dose rat studies, sitosterol and sitostanol were found in the adrenals, ovary and stomach at low concentrations, campestanol in the adrenals, ovaries and intestinal epithelia, and campesterol in the adrenals, spleen, intestinal epithelia, ovaries, liver and bone marrow. Excretion is via the faeces as both free sterol and sterol esters (SCF, 2003; ANZFA, 2001).

The toxicokinetics of sterol esters after oral uptake are comparable to those of sterols as they are hydrolysed to free sterols in the intestine as part of the normal digestive process (ANZFA, 2001).

No information could be found on absorption via the dermal route. However, given their high octanol/water partition coefficient (log Pow >> 8, see Section 1.3), sterols and sterol esters are not expected to be taken up through skin to a significant degree (as per Table R 7.12-3, REACH Endpoint specific guidance R.7c of May 2008).

Squalene:

Animal studies indicate that squalene is poorly absorbed from the gastro-intestinal tract and slowly absorbed through the skin. Absorption occurs through the lymphatic vessels (similar to cholesterol), with partial cyclization to sterols during the transit through the intestinal wall. Absorbed squalene is preferentially converted to bile acids in the liver (CIR, 1982).

Little information could be found on absorption via the dermal route. However, given its high log Kow >> 6, squalene is not expected to be taken up through skin to a significant degree (as per Table R 7.12 -3, REACH Endpoint specific guidance R.7c of May 2008). This is in line with what was seen in a study by Butcher (1951) who saw little evidence that squalene penetrated through the skin of rats when applied dermally.

Hydrocarbons:

Absorption of alkanes may occur through the portal and/or the lymphatic system. For n- and cycloalkanes the oral absorption varies from 90% for C14-C18 to 25% for C26-C29. The absorption further decreases with increasing carbon number, until above C35 when it is negligible. Limited data suggest that cyclo-alkanes are absorbed at similar levels as n-alkanes of comparable molecular weight, whereas absorption of branched alkanes is slightly less (EFSA, 2012). Alkanes are initially oxidised to the corresponding fatty alcohols by the cytochrome P450 system, subsequently biotransformed to fatty acids and in some cases subjected to the normal β-oxidation pathway. This reaction is more rapid for n-alkanes than for branched- and cyclo-alkanes. Due to low biotransformation rates, in particular for some branched- and cyclo-alkanes, Mineral oil saturated hydrocarbons (MOSH) having carbon number between 16 and 35 may accumulate in different tissues including adipose tissue, lymph nodes, spleen and liver. In rats, the terminal half-life of MOSH in blood (estimated from P15(H) white oils) was between 23 and 59 h, depending on the strain. However, this reflects the elimination of the easily degraded MOSH. The concentration of MOSH in human tissues (mainly lymph nodes, liver, spleen and adipose tissue) demonstrates that accumulation of these compounds, mostly branched- and cyclo-alkanes, occurs in humans (EFSA, 2012).

With regard to the dermal route, studies with humans, animals, and excised skin have all shown that C5–C12 alkanes are poorly absorbed following dermal administration (Mckee et al., 2015).

Cross-reference 3

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

The repeated dose toxicity potential of the test substance ‘squalene-rich fraction obtained from vegetable oil deodorizer distillate by transesterification, crystallisation and vacuum distillation’ can be deduced based on information available for its individual constituents. Studies conducted with the major constituents, glycerides, fatty acids and unsaponifiable matters (including tocopherols, sterols, squalene and hydrocarbons) indicate overall low toxicity. The sterols, sterol esters and squalene are found to be poorly absorbed both orally and dermally, therefore unlikely to contribute significantly to toxicity. Chronic and sub-chronic repeated dose studies with sterols and squalene identified NOAELs at 2500 and 3300 mg/kg bw/day respectively. Tocopherols, on the other hand, were well absorbed and although beneficial at nutritionally relevant doses, may antagonise the function of other fat-soluble vitamins at higher doses. Further, several safe limits (in the form of ULs or ADIs) have been derived for tocopherols by the different regulatory authorities (SCF/EFSA, IOM, EVM). Considering the overall low inherent toxicity potential of the constituents, the latest EFSA re-evaluated upper limit based on a NOAEL of 540 mg/day or 7.7 mg/kg bw/day for a 70 Kg adult, has been considered further hazard and risk assessment.

Considering the intermediate use and physico-chemical properties of the test substance and/or its constituents, the absorption potential via the dermal route is not expected to be higher than oral route. Exposure via the inhalation route is not expected considering the intermediate use and low vapour pressure of the substance. Therefore, testing via these routes is unlikely to result in any additional hazard identification and hence further testing involving vertebrate animals may be omitted, in accordance with Annex XI (1.2) of the REACH regulation. The risk assessment for the likelihood of any potential dermal or inhalation exposure in workers has been conducted based on the conservative NOAEL identified from the studies with the constituents and by using appropriate route-to-route extrapolation assessment factors as per the ECHA Guidance R.8.

Toxic effect type:

dose-dependent

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

7.7 mg/kg bw/day

Study duration:

chronic

Experimental exposure time per week (hours/week):

168

Species:

other: humans

Quality of whole database:

all studies with constituents were taken from peer reviewed or regulatory opinions; NOAEL based on the EFSA re-evaluated upper limit for tocopherol

Endpoint conclusion:

no study available

Endpoint conclusion:

no study available

Endpoint conclusion:

no study available

Endpoint conclusion:

no study available

In the absence of repeated dose toxicity study with the test substance,‘squalene-rich fraction obtained from vegetable oil deodorizer distillate by transesterification, crystallisation and vacuum distillation’ the endpoint has been assessed based on animal and human studies for substances representative of the main constituents, which can be categorised as glycerides, fatty acids or fatty acid methyl esters (which will eventually hydrolyse to fatty acids (Mattson and Volpenhein, 1972)) and unsaponifiable matters (including tocopherols, sterols, squalene and hydrocarbons). As a large number of studies have been conducted on the individual constituents, particularly in the context of nutritional research, for practical reasons, only a limited number of studies are reported here.

Oral:

Glycerides and fatty acids:

At doses ranging from 7.5 to 18.5% in the diet, no significant toxicity was seen for various constituent glycerides and fatty acids with chain lengths varying between C8-18 or C16-18, including C18-unsatd. and C18-unsatd. hydroxy (Morin, 1967; Harkins and Sarrett, 1968; Nolen, 1981; Manorama and Rukmini, 1991; Coquet et al., 1977; Speijers et al., 2009; Irwin, 1992; HERA, 2002). The highest oral NOAEL could therefore be considered to be 18.5% in diet, i.e., approximately 9,250 mg/kg bw/day. In certain studies (also others not reported here), differences may be observed compared to controls on bodyweight gain, food consumption and certain measured parameters depending on the chain length distribution of the fatty acids (associated to the glycerides or free) and their degree of unsaturation. However, research indicates that when consumed at nutritionally relevant concentrations, there are no adverse effects on health and longevity. It is worth noting that, due to their innocuous nature, fats and oils are commonly used as controls and as vehicles in animal toxicity studies. For example, OECD Guideline 408 (repeated dose 90-day oral toxicity study in rodents) recommends the use of “a solution/emulsion in oil (e.g. corn oil)” as a vehicle where an aqueous vehicle is not suitable (OECD, 1993). Several fatty acids (stearic acid; oleic acid and sodium palmitate) are Generally Recognised as Safe (GRAS) by the U.S. Food and Drug Administration (US FDA). Also, fatty acids as a group are permitted as direct food additives (HERA, 2002).

Unsaponifiable matter:

Tocopherols:

Tocopherols have been tested in numerous repeated dose oral toxicity studies. Overall, animals appear to tolerate high levels (i.e. at least two orders of magnitude above nutritional Vitamin E levels, e.g. 1,000 - 2,000 IU/kg diet) without adverse effects. At very high doses, signs indicative of antagonism with the function of other fat-soluble vitamins occur (Tomassi and Silano, 1986; Fiume, 2002; EFSA, 2008). A NOAEL of 125 mg/kg bw/day was reported for a 64-week feeding study in rat with tocopheryl acetate (Tomassi and Silano, 1986).

The tocopherols and their stereoisomers have also been reviewed and evaluated by a number of regulatory authorities along with derivation of the threshold/safe upper limits: 

1)   A group ADI of 0.15–2 mg/kg bw/day for dl-α-tocopherol and d-α-tocopherol was allocated by Joint FAO/WHO Expert Committee on Food Additives (JECFA) in 1987. This is based on clinical studies in humans and takes into account the fact that α-tocopherol is an essential nutrient (JECFA, 1987).

2)   The Antioxidant Panel of the Food and Nutrition Board (FNB) at the Institute of Medicine of the US National Academy of Sciences set the tolerable upper intake level (UL) for adults at 1000 mg (2,325 μmol)/day of any supplemental form of alpha-tocopherol. This was based on a LOAEL of 500 mg/kg bw/day for dl-α-tocopherol (based on reduced blood clotting in thestudy by Wheldon et al. (1983)), divided by an uncertainty factor of 36 (LOAEL to NOAEL = 2; subchronic to chronic intake = 2; intra-species variation = 3; inter-species variation = 3) and assuming a human body weight of 68.5 kg. The same UL for pregnant and lactating women was recommended. For infants (0–12 months), a UL was not determined, as it was considered that the only source of intake should be from food or formula. The recommended ULs for ages 1–3, 4–8, 9–13 and 14–18 years were 200, 300, 600 and 800 mg/day of any isomeric form of α-tocopherol, respectively (IOM, 2000).

3)   The EU Scientific Committee on Food (SCF) has not derived an ADI for vitamin E or any of the tocopherols, but based on a placebo controlled, dose-response supplementation study in 88 healthy humans (Meydani et al., 1998), set the NOAEL at 540 mg alpha-tocopherol equivalents, the highest dose used in the study. Considering an uncertainty factor of 2 to cover for interindividual differences in sensitivity, the SCF established an UL of 270 mg alpha-tocopherol equivalents for adults, which was rounded to 300 mg alpha-tocopherol equivalents, which is equivalent to 5 mg/kg bw/day in a 60 kg adult. The ULs were scaled for children in the age ranges 1–3, 4–6, 7–10, 11–14 and 15–17 years to give ULs of 100, 120, 160, 220 and 260 mg/day, respectively (SCF, 2003).

4)   The UK’s Expert Group on Vitamins and Minerals (EVM) established a NOAEL for humans (age not defined) of 540–970 mg d-α-tocopherol equivalents per day (EVM, 2003). An uncertainty factor to account for inter-individual differences was not considered necessary, as human study results (such as those of Meydani et al. (1998)) supported a NOAEL of 540 mg d-α-tocopherol equivalents per day; therefore, the safe upper limit was established as 540 mg d-α-tocopherol equivalents for supplemental vitamin E, which is equivalent to 90 mg/kg bw/day in a 60 kg adult (EVM, 2003).

5)   The EFSA Scientific Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food (AFC) reviewed and concluded that mixed tocopherols (d-α-tocopherol, d-β-tocopherol, d-γ- tocopherol and d-δ-tocopherol) are not a safety concern at the proposed levels of use of 300 mg/day or 5 mg/kg bw/day for 60 kg adults (EFSA, 2008).

 

Sterols and sterol esters:

In animal studies, sterols and sterol esters demonstrated low repeated dose oral toxicity. The lowest NOAEL found was equivalent to ca. 2,500 mg sterols/kg bw/day in a 22-month study with rats (SCF, 2003). Healthy human volunteer studies conducted with phytosterols at 13-47 mg sterols/kg bw/day in diet, revealed no significant systemic toxicity other than minor adverse effects such as moderate constipation, mildly increased body weights, deficiencies of carotenoid and fat soluble (Vit E and K1) vitamin levels etc. These deficiencies of the carotenoid and vitamin levels were not reported to be associated with any symptoms and the highest tested dose was well tolerated. Furthermore, phytosterols are also well known to reduce the serum cholesterol levels and their efficacy depends on the dose, the existing serum cholesterol level and the nature of the phytosterols ingested. Apart from carotenoid-lowering effects, no other adverse effects were observed in humans with repeated intake as high as 8 g/day, although a maximum uptake of 3 g/day (i.e. 50 mg/kg bw/day for a 60 kg adult) is recommended, with no added benefits observed above that level (SCF, 2002).

Squalene:

A study was conducted to determine the effect of co-administration of squalene in sub-chronic repeated dose study by oral administration. The general plan of the experiment was to feed two groups of rats on a complete artificial diet, while one group received in addition a small quantity of squalene each day. Immediately before feeding-time the animals were given approximately 660 mg of squalene dropped directly into their mouths from a micro-burette. Records were taken of the amount of squalene given each day. The faeces were collected throughout the experiment at weekly intervals and stored in alcohol. At the end of the experiment the animals were killed by chloroform and their livers removed as free from blood as possible. No obvious ill effects followed the administration of squalene and postmortem examination showed that the organs of the animals were very healthy. There appeared to be a greater deposition of fat in the animals which received squalene and on the whole they seemed to grow at a somewhat greater rate than those of the control groups, possibly because the laxative action of the squalene caused a greater food consumption. The experiment showed that when squalene was administered to the rat, it was in part absorbed and as a result of absorption there was a marked increase in the amounts of unsaponifiable matter and of cholesterol in the body and liver of the animal. No effects up to dose of 3300 mg/kg bw/day (considering 200 g average animal body weight and 660 mg per animal dose) squalene in any animals were reported. Under the study conditions, the NOAEL of squalene was determined to be 3300 mg/kg bw/day in rats (Channon, 1926).

Apart from the presented study and the epidemiological evidence deriving from the presence of squalene as a component of common edible oils and fat constantly present in human diet, several studies have been performed in the framework of use of squalene as adjuvant in recently developed influenza vaccines. The evaluation report for Humenza (vaccine against H1N1 virus containing squalene-based adjuvant) submitted to the European Medicines Agency (EMA) in 2010, reported two repeat dose toxicity studies, one in rodents (in rats) and one in non-rodents (in rabbits) for assessing systemic toxicity and local tolerance. In the rat repeat dose and in the reproductive toxicity studies, several dose levels of the vaccine were assessed (from 1.25% to 10% squalene, including the human dose 2.5% squalene). In the rabbit repeat dose toxicity study, the human dose of the squalene-based adjuvant used in the vaccine was evaluated. No specific safety concerns were raised. Similar toxicology studies were submitted by Novartis, which were designed to meet the US FDA and EMEA requirements complying with applicable international guidelines for the nonclinical assessment of vaccines and adjuvants. The pivotal toxicology studies performed with the adjuvant alone included the evaluation of local tolerability, repeat-dose toxicity (one clinical dose administered to rabbits once daily for 14 days), genotoxicity, sensitisation, and embryofetal and developmental toxicity. These studies did not indicate any potential for systemic toxicity or local reactogenicity. In repeat-dose rabbit studies, clinical pathology findings of increased fibrinogen, and minor inflammatory and degenerative changes at the injection site, which were consistent with the effects of intramuscular (i.m.) injections of an immunological adjuvant. These reactions were readily reversible within days to 1–2 weeks. Furthermore, squalene is a common component of the human diet through olive oils and other oils and fats and it is assumed in a quantity between 30 and 200 mg/day, without adverse effect in a whole human life. It is also used since decades in cosmetic formulations, creams and similar, where a daily application is performed during a lifetime by humans. No systemic effects have been reported, about eventual systemic toxicity.

Hydrocarbons:

In a subchronic dietary toxicity study, six highly refined base oils were administered to male and female F-344 rats at dose levels 20, 200, 2,000, and 20,000 ppm of for 90-days. High-dose dietary exposure produced a syndrome of effects in liver and mesenteric lymph nodes with response greater in females than in male F-344 rats. Effects include increased organ weights, evidence for the presence of nonpolar hydrocarbons, and other observations suggestive of an inflammatory response. A NOEL of 20 ppm or 2 mg/kg bw/day for 4 of 6 oils (N10A, P15H, N70A, N70H; carbon number ranging from 15 to 37) was calculated, a NOEL was not established for N15H (carbon number 17-30); and a NOEL of greater than or equal to 20,000 ppm or 1900 mg/kg bw/day was calculated for P100H (carbon number 28-45); based on histiocytosis. In another sub-chronic dietary toxicity study for P15(H) white oil (carbon number 18-30), the NOAEL in SD female rats and LOAEL in F344 female rats were determined to be ≥1624 mg/kg bw/day (based on absence of adverse effects) and 161 mg/kg bw/day (based on the histopathology in the liver and mesenteric lymph nodes accompanied by changes in the weight of those organs) respectively (Firriolo, 1995). As these effects are now shown to be strain-specific, the adversity of the mesenteric lymph nodes (MLN) histiocytosis is questionable. In two-year dietary studies conducted to determine the chronic toxicity and the carcinogenicity of P70(H) (carbon number 27-43) and P100(H) (carbon number 28-45) white mineral oils in Fischer-344 rats (F-344), the NOAEL for these studies was considered to be 1200 mg/kg bw/day (highest tested dose), based on the absence of carcinogenicity and chronic toxicity (Trimmer, 2004).

Based on the available weight of evidence information on the constituents, the test substance ‘squalene-rich fraction obtained from vegetable oil deodorizer distillate by transesterification, crystallisation and vacuum distillation’ is overall considered to have low systemic toxicity.

Inhalation:

There is no information on the repeated dose inhalation toxicity of the test substance. However, exposure via the inhalation route is not expected considering the intermediate use and low vapour pressure (0.00108 Pa at 20°C) of the substance. In addition, studies conducted via the oral route with the constituents or its representative substances indicated a lack of significant toxicity of the test substance. Therefore, testing via inhalation route is unlikely to result in any additional hazard identification and hence further testing involving vertebrate animals may be omitted, in accordance with Annex XI (1.2) of the REACH regulation. The risk assessment for the likelihood of any potential inhalation exposure in workers has been conducted based on the conservative NOAEL identified from the studies with the constituents and by using appropriate route-to-route extrapolation assessment factors as per the ECHA Guidance R.8.

Dermal:

There is no information on the repeated dose dermal toxicity of the test substance. However, given the intermediate use of the test substance where it completely gets converted to crude squalene, there is only a low dermal exposure potential of the test substance itself in workers, during its handling. However, as discussed in the toxicokinetic section, dermal absorption of the test substance is not expected to be higher than via the oral route. Considering the intermediate use and physico-chemical properties of the test substance and/or its constituents, the absorption potential via the dermal route is not expected to be higher than oral route. Therefore, testing via the dermal route is unlikely to result in any additional hazard identification and further testing involving vertebrate animals may be omitted, in accordance with Annex XI (1.2) of the REACH regulation. The risk assessment for the likelihood of any potential dermal exposure in workers has been conducted based on the conservative NOAEL identified from the studies with the constituents and by using appropriate route-to-route extrapolation assessment factors as per the ECHA Guidance R.8.

Based on the available weight of evidence information on the constituents, the test substance ‘squalene-rich fraction obtained from vegetable oil deodorizer distillate by transesterification, crystallisation and vacuum distillation’ does not warrant classification for repeated dose toxicity according to EU CLP criteria (EC 1272/2008).

Results and discussion

Target system / organ toxicity

Critical effects observed:

not specified