Registration Dossier

Administrative data

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Literature study well described and of enough reliability to assess the endpoint

Data source

Reference
Reference Type:
publication
Title:
Genotoxicity assessment of vaccine adjuvant squalene
Author:
D. Yüzbasıog˘lu et al.
Year:
2013
Bibliographic source:
Food and Chemical Toxicology 56 (2013)240–246

Materials and methods

Principles of method if other than guideline:
Assay, approximately 100 ul whole blood was collected from rat’s tail vein into lithium–heparintubes. 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 (Smith et al., 2008 ). 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 described above was applied
GLP compliance:
not specified
Type of assay:
mammalian comet assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
The test substance squalene (minimum98% purity) was purchased from Sigma and dissolved in physiological saline

Test animals

Species:
rat
Strain:
Wistar
Details on test animals 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
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 / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0.07 mg/Kg
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0.14 mg/Kg
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0.28 mg/Kg
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0.56 mg/Kg
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
1.12 mg/Kg
Basis:
nominal conc.
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
Sex:
not specified
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative