Registration Dossier

Administrative data

Endpoint:
specific investigations: other studies
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2009
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Report describes methodology employed to try to measure genotoxicity using the Comet Assay in nasal epithelial tissue in rats.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
Study required under (EC) No 466/2008
GLP compliance:
no
Type of method:
in vivo
Endpoint addressed:
genetic toxicity

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
No test material was used. The method development is described.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Not specified

Administration / exposure

Route of administration:
other: Not applicable
Vehicle:
other: Not applicable
Details on exposure:
Naive animals were used for method development.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
Not applicable
Duration of treatment / exposure:
Not applicable
Frequency of treatment:
Not applicable
Post exposure period:
Not applicable
Doses / concentrations
Remarks:
Doses / Concentrations:

Basis:
other: Not applicable - naive animals only
No. of animals per sex per dose:
9 animals
Control animals:
no
Details on study design:
Various enzymatic digestion procedures were used to isolate single cells from the nasal and tracheal epithelium including:

digestion with collagenase type IV for 30 or 40 min at 37°C or overnight at 4°C; combined digestion with collagenase type IV / collagenase type P and protease type XIV for 45 min at 37°C.

The resultant preparations were washed and cells recovered using centrifugation.

Cell viability as indicated by means of trypan blue exclusion was typically 60% to 83%. This compares with recent publications which recommend a minimal viability of at least 70% for the in vivo Comet Assay.

The number of viable cells recovered per animal from nasal epithelium (1-3.5x106 cells) and trachea (no detectable cells recovered) was less than expected. The yield of individual cells from nasal epithelium, while low, was nonetheless considered adequate for subsequent conduct of the Comet assay; the very low recovery from tracheal epithelium precluded further work.

The number of blood cells present was reduced by including additional washing steps.

Single cell gel electrophoresis revealed the presence of extensive DNA damage in nasal epithelial cells expressing better than 70% viability as assessed by trypan blue exclusion. In some cases bright background fluorescence was seen after ethidium bromide staining. In general only around 10% of the cells applied to the gel exhibited an intact DNA image, with a single (exceptional) preparation of cells showing around 50% intact DNA.

Examinations

Positive control:
Not applicable

Results and discussion

Details on results:
Several approaches were taken in order to obtain viable cell populations from the nasal turbinates of rats for the purposes of establishing a method for Comet Assay determination on the TBHP test material.

Viability ranged from 60-83% (acceptable criterion: 70%)
Hedgehogs: 44-49% (acceptable criterion = 20%)
Tail intensities: 13.2, 8.3 (acceptable criterion = 5)

Applicant's summary and conclusion

Conclusions:
The Comet Assay method was not successful in nasal epithelial tissues from the rat, due to unacceptable non-specific cell DNA damage and low cell viability.
Executive summary:

An attempt was made to develop a method to generate local nasal epithelial tissue genotoxicity data using the Comet Assay in the rat. Through multiple attempts and variations on published methods, it was not possible to obtain cell suspensions of with viability and DNA stability which fell within acceptable quality standards from which to make an assessment of the hazards/risks of locally acting genotoxic agents in the nasal tissue.