Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
data waiving: supporting information
Reference

The vapour pressure of the test substance was determined using the isothermal thermogravimetrical effusion method according to OECD Guideline 104 and EU Method A.4 (Wohr, 2019).

Vapour pressure:
0.001 Pa
at the temperature of:
20 °C
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
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).

Data source

Materials and methods

Results and discussion

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

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