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

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

Comet assay

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

publication

Title:

Genotoxicity assessment of vaccine adjuvant squalene

Author:

Yüzbaşıoğlu D et al

Year:

2013

Bibliographic source:

Food Chem Toxicol. 2013 Jun;56:240-6

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

The in vivo DNA damaging potential of squalene was evaluated in rats following sub-cutaneous injection and quantification of tail length and tail intensity.

GLP compliance:

not specified

Type of assay:

mammalian comet assay

Test material

Test material information

Constituent 1

Reference substance name:

2,6,10,15,19,23-hexamethyltetracosa-2,6,10,14,18,22-hexaene

EC Number:

203-826-1

EC Name:

2,6,10,15,19,23-hexamethyltetracosa-2,6,10,14,18,22-hexaene

Cas Number:

111-02-4

Molecular formula:

C30H50

IUPAC Name:

2,6,10,15,19,23-hexamethyltetracosa-2,6,10,14,18,22-hexaene

Details on test material:

- Name of test material (as cited in study report): Squalene

Test animals

Species:

rat

Strain:

Wistar

Sex:

not specified

Details on test animals or test system and environmental conditions:

Wistar rats (10–12weeks old) were procured from RefikSaydam National Public Health Agency, Experimental Animal Unit (Ankara, Turkey).Rats were kept in separate cages in an experimental room under controlled conditions of temperature (22±2 C) and humidity (50– 60%) with feed and water being available ad libitum. Lighting was controlled to provide 12 h artificial light followed by 12 h darkness.

Administration / exposure

Route of administration:

subcutaneous

Vehicle:

- Vehicle(s)/solvent(s) used: None

Duration of treatment / exposure:

Group 1 was the treatment group studied 1day after the squalene injection.
Group 2 was studied 14 days after squalene injection

Frequency of treatment:

Single

Post exposure period:

1 and 14 days

Doses / concentrations

Remarks:

Doses / Concentrations: 0, 0.07, 0.14, 0.28, 0.56, 1.12 mg/kg bw
Basis: nominal concentration

No. of animals per sex per dose:

5 animals per dose

Control animals:

yes, concurrent no treatment

Positive control(s):

yes, with mitomycin-C 2 mg/Kg

Examinations

Evaluation criteria:

Quantification of DNA breakage was realized using Comet Image Analysis System (‘‘Comet Assay IV’’, Perceptive Instruments Ltd., UK).At least 300 comets for each experimental group were recorded as tail length and tail intensity.

Statistics:

For data evaluation, the z-test was used for the percentage of abnormal cells, CA/cell, MI, RI, NDI, MN assays. The t-test was applied for SCEs and comet assay results to determine the statistical difference between treated and untreated samples. Dose–response relationships were determined from the correlation and regression coefficients for the percentage of abnormal cells, CA/cell, SCE, mean MN and DNA.

Results and discussion

Test results

Key result

Sex:

not specified

Genotoxicity:

positive

Toxicity:

no effects

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

DNA damage was seen in lymphocytes of rats one day after injection of squalene in in vivo (Group 1). Squalene significantly increased Comet Tail Length (CTL) at 0.14, 0.28, 0.56 mg/kg doses however it significantly decreased CTL at 0.07 and 1.12 mg/kg doses. Whereas, squalene decreased comet tail intensity (CTI) at all doses (except 0.28 mg/kg), however, this decrease was significant only at 0.07 and 1.12 mg/kg doses compared to control. In contrast to the results obtained for group 1, in group 2, squalene significantly increased both CTL and CTI (except 0.07 mg/kg) at all doses used but 1.12 mg/kg.

Any other information on results incl. tables

For result tables, kindly refer to the attached background material section of the IUCLID.

Applicant's summary and conclusion

Conclusions:

Under the test conditions, the substance was considered to be genotoxic in an in vivo comet assay in rat.

Executive summary:

A study was performed to investigate the genotoxic potential of the test substance, squalene using in vivo mammalian comet assay in rat. Five dose levels of squalene, determined based on the amount of squalene in human vaccines (0.07 mg/kg, 0.14 mg/kg, 0.28 mg/kg, 0.56 mg/kg, 1.12 mg/kg) were given to rats subcutaneously for each treatment groups (5 animals/groups). In addition, an untreated control and a positive control (mitomycin-C,2mg/kg) were also used to test the validity of the assay for all treatment groups. Rat blood samples taken from two different groups were used in in vivo Comet assay. Group 1 was the treatment group studied 1 day after the squalene injection. Group 2 was studied 14 days after squalene injection. For in vivo comet assay, approximately 100 µL whole blood was collected from rat’s tail vein into lithium–heparin tubes. Due to the intensity of cells, blood samples were diluted and suspended with phosphate buffer (pH:7.4) in 1:1 ratio and then centrifuged. Lymphocytes were isolated by Biocoll separating solution. After the isolation step, lymphocytes were resuspended in PBS (phosphate buffered saline). Afterwards, the protocol for in vitro comet assay was applied. Quantification of DNA breakage was realized using Comet Image Analysis System. At least 300 comets for each experimental group were recorded as tail length and tail intensity. DNA damage was seen in lymphocytes of rats one day after injection of squalene in in vivo (Group 1). Squalene significantly increased Comet Tail Length (CTL) at 0.14, 0.28, 0.56 mg/kg doses however it significantly decreased CTL at 0.07 and 1.12 mg/kg doses. Whereas, squalene decreased comet tail intensity (CTI) at all doses (except 0.28 mg/kg), however, this decrease was significant only at 0.07 and 1.12 mg/kg doses compared to control. In contrast to the results obtained for group 1, in group 2, squalene significantly increased both CTL and CTI (except 0.07 mg/kg) at all doses used but 1.12 mg/kg. Under the test conditions, the substance was considered to be genotoxic in an in vivo comet assay in rat (Yüzbaşıoğlu, 2013).