Registration Dossier

Administrative data

Description of key information

NOAEL oral rat > 660 mg/Kg bw

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1926
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Good publication
Qualifier:
no guideline followed
Deviations:
not applicable
Principles of method if other than guideline:
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 for 42 days
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
not specified
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Diet: complete artificial diet that described by Drummond and Coward [1920].
Route of administration:
other: dropped directly into their mouths from a micro-burette.
Vehicle:
not specified
Duration of treatment / exposure:
dropped directly in mouths from a micro-burette.
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
660 mg
Basis:
actual ingested
No. of animals per sex per dose:
test group: 10 rats
negative control: 9 rats
Control animals:
yes, concurrent no treatment
Observations and examinations performed and frequency:
Treatment of livers:
At the end of the experiment the animals were killed by chloroform and their livers removed as free from blood as possible.

Treatment of faeces:
The faeces were stored in alcohol.
Other examinations:
The faeces were collected throughout the experiment at weekly intervals and stored in alcohol.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Details on results:
OTHER FINDINGS

It is clear from the weight of unsaponifiable matter from the faeces of the latter animals, and from its iodine value, that much of the squalene administered has been excreted.
Dose descriptor:
NOAEL
Effect level:
ca. 660 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: body weight; amounts of cholesterol,
Critical effects observed:
not specified

If we assume that that fraction of this unsaponifiable matter which is not squalene has an iodine value which is the same as that of the material from the control group, namely 80.4%, then we may calculate from the iodine value of the whole of this unsaponifiable matter (316), that the amount of squalene present is 81.41 %, i.e. 16 -79 g. (squalene has iodine value 371). This figure can only be approximate, but some rough check on it is obtained from the fact that, if we subtract the weight of material not sterol in the faeces of the control group from the corresponding figure for the faeces of the animals receiving squalene, we arrive at the figure 17-60 g. These figures 16.79 and 17.60 g. can only be approximate, but they would seem to show that about 17 g. of the 27.7 g. of squalene administered had been excreted. Thus about 10 g. have been absorbed during the course of 42 days and this absorption has resulted in a marked increase in the weight of unsaponifiable matter in the livers and also in the bodies of these animals, namely, from 0.3680 to 1.0774 g. in the case of the livers and from 1.8620 to 2 .9114 g. in that of the bodies. There is also a large increase in the amounts of cholesterol, from 0.2576 to 0.6189 g. in the livers and from 1.3000 to 2.1620 g. in the bodies.

No obvious ill effects followed the administration of squalene and post mortem 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.

Conclusions:
The experiment shows that when squalene is administered to the rat, it is in part absorbed and as a result of absorption there is a marked increase in the amounts of unsaponifiable matter and of cholesterol in the body and liver of the animal. No effect up to the dose of 3300 mg/Kg bw has been reported.
Executive summary:

Squalene has been tested in subchronic repeted 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. The experiment shows that when squalene is administered to the rat, it is in part absorbed and as a result of absorption there is a marked increase in the amounts of unsaponifiable matter and of cholesterol in the body and liver of the animal.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
660 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The database of accessible data is poor, but squalene has been extensively studied as vaccin adjuvant and several references can be cited that conferms the result. 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

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

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 influence vaccins.

 

In 2010, the European Medicines Agency (EMA) has reviewed a vaccine against the h1n1 influenza virus, called “Humenza” which contained a, then novel, squalene based adjuvant (AF03, developed and studied by Glaxo Smithkline). The squalene content in AF03 is 12.4 mg and the risk-benefit balance of the influenza vaccine was considered to be favourable in a pandemic situation.

The evaluation report for "Humenza" among others mentioned that there are other squalene oil-in-water adjuvanting emulsions licensed in other influenza vaccines (e.g. AS03 and MF59). In the evaluation is reported that two repeat dose toxicity studies, one in rodents (in rats: AA32695) and one in non rodents (in rabbits: AA33212), assessed the systemic toxicity and local tolerance of AF03. In the rat repeat dose and in the reproductive toxicity studies, several dose levels of AF03 were assessed (from 1.25% to 10% squalene, including the human dose 2.5% squalene) in order to establish a dose effect relationship and determine the no observed adverse effect level (NOAEL). In the rabbit repeat dose toxicity study, the human dose of AF03 used in the vaccine was evaluated. No specific safety concerns were raised. The adjuvant MF59 has been studied by Novartis and some reviews have been published on the presented studies. In the review "Safety of MF59 adjuvant", (Schultze and al., 2008) the author reported that MF59TM administered alone and in combination with a variety of antigens has been tested in several animal models including mice (product release, immunogenicity, challenge, and micronucleus test), rats (reproductive toxicity), Guinea pigs (product release, immunogenicity, and sensitisation), rabbits (immunogenicity, standard toxicology, and reproductive toxicity), dogs (toxicology), goats (immunogenicity) and several non-human primates, including chimpanzees (immunogenicity and efficacy testing). The regulatory toxicology studies (internal reports) with MF59TM and MF59TM-adjuvanted antigens were designed to meet United States Food and Drug Administration and the European Medicines Evaluation Agency requirements and complied with applicable international guidelines for the nonclinical assessment of vaccines and adjuvants. The pivotal toxicology studies performed with MF59TM 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 provided the basis for using MF59TM as an adjuvant platform for combination with many antigens. The nonclinical safety program, using several animal species, provides a complete and accurate assessment of the safety of MF59TM for use as an adjuvant. Its use is not associated with any potential for systemic toxicity and it has a loworder of local reactogenicity. In repeat-dose rabbit studies, clinical pathology findings of increased fibrinogen, and minor inflammatory and degenerative changes at the injection site are consistent with the effects of intramuscular (i.m.) injections of an immunological adjuvant. These reactions are readily reversible within days to 1–2 weeks.

Finally, squalene has been used since decades in cosmetic formulations, creams and similar, where a daily application is performed during a lifetime by humans. No effects have ever been reported, about eventual systemic toxicity.

 

As a conclusion the registrant is considering Squalene not toxic for repeated dose toxicity and he is not proposing any further test on repeated dose toxicity .


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Public study

Justification for classification or non-classification

The substance is not classified orally toxic because it doesn't meet the classification criteria of the CLP regulation n. 1272/2008 on repeated exposure:

Category 1: substances that have produced significant toxicity in humans or that, on the basis of evidence from studies in experimental animals, can be presumed to have the potential to produce significant toxicity in humans following repeated exposure.

Category 2: substances that, on the basis of evidence from studies in experimental animals can be presumed to have the potential to be harmful to human health following repeated exposure.

For significant toxicity the DMEL coming from subacute studies is general considered lower than 300 mg/Kg bw. In all presented studies the subacute DMEL is well above 300 mg/Kg bw