Registration Dossier

Administrative data

in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From December 2nd, 2011 to February 1st, 2012
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
equivalent or similar to
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Version / remarks:
First OECD Guideline published on 26 September 2014, but same criteria were followed in the study
GLP compliance:
Type of assay:
mammalian comet assay

Test material

Test material form:
solid: particulate/powder

Test animals

Details on test animals and environmental conditions:
- Source: Charles River Italia S.p.A., Calco (Lecco) Italy
- Age at study initiation: 8-10 weeks
- Weight at study initiation: approximately 300 grams
- Housing: 5 animals/cage (4 animals/cage for the positive control group and 2 animals/cage for the preliminary toxicity assay), in polycarbonate cages with a stainless steel mesh lid and floor
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

- Temperature: 22 °C ± 3 °C
- Humidity: 55 % ± 15 %
- Photoperiod: 12 hour light/dark period

Administration / exposure

Route of administration:
oral: gavage
- Vehicle used: sterile distilled water of injectable grade
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: solutions of the test item were prepared in sterile distilled water of injectable water.
Duration of treatment / exposure:
2 days
Frequency of treatment:
Animals were dosed twice, at 24 hour interval.
Post exposure period:
3-4 hours
Doses / concentrationsopen allclose all
Dose / conc.:
2 000 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
Dose / conc.:
500 mg/kg bw/day
No. of animals per sex per dose:
5 animals per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
Ethylmethanesulphonate (EMS) prepared in injectable water
- Route of administration: oral gavage
- Doses / concentrations: 200 mg/kg bw/day
- No. of animals of positive group: 4 animals


Tissues and cell types examined:
Peripheral blood and cell suspensions isolated from liver and jejunum/ileum.
Details of tissue and slide preparation:
Dose levels for main experiment were selected based on a preliminary toxicity experiment where the test item was administered twice by oral gavage to groups of two male and two female rats to confirm survival at the dose levels of 2000 and 1000 mg/kg bodyweight.

The animals were sacrificed at the appropriate time after treatment by asphyxiation with carbon dioxide. Prior to sacrifice blood was taken from the tail vein.
From each animal, a section of liver was removed, cut into 3 or more pieces and gently agitated in a Petri dish full of ice-cold phosphate buffered saline (PBS) containing 20 mM EDTA and 10 % DMSO (mincing solution) to remove as much blood as possible. The sample was placed into 8 ml of ice-cold mincing solution and minced into finer pieces. The cell suspension was filtered through a 100 µm gauze and centrifuged at 500 rpm for 10 minutes at approximately +4 °C.
The pellet was resuspended in ice-cold PBS and the cell concentration was adjusted to a final concentration of 1x10^6 cells/ml using PBS and used for slide preparation.
A portion of jejunum and ileum was removed from each animal, cleaned in a Petri dish full of ice-cold mincing solution and irrigated with this ice-cold solution using a Pasteur so the remaining food and cellular debris could run down to waste. The portion of jejunum and ileum was transferred to a clean Petri dish and opened. The cell layer was gently scraped using a cell scraper and placed in 5 ml of ice-cold mincing solution. The cell suspension was filtered through a 100 µm and a 70 µm gauze and centrifuged for 8 minutes at approximately +4 °C. In order to better collect the cell suspension, centrifugation was performed for most animals at 1200 rpm and not 1000 rpm.
The pellet was resuspended in 500 µl of ice-cold PBS; cells were counted at the microscope using a haemocytometric chamber and concentration adjusted when possible at approximately 1x10^6 cells/ml by adding PBS.

For each cell suspension, 60 µl was added to 390 µl of 0.7 % (w/v) low melting point agarose (LMA), pre-warmed at approximately 37 °C.
For peripheral blood, 30 µl of whole blood was taken from the tail vein and added in a vial containing 450 µl of 0.7 % (w/v) LMA.
An aliquot of 75 µl of each cell suspension was dispensed onto glass microscope slides precoated with a layer of 1 % (w/v) normal melting point agarose (NMA).
Four slides per tissue were prepared.
The slides were covered with a clean coverslip and the gels were allowed to solidify. The coverslips were then removed and a further 75 µl of LMA added over the cell layer and covered with a clean coverslip. The gels were allowed to solidify and the coverslips were carefully removed. The slides were immersed in freshly prepared pre-cooled lysis solution and left overnight at approximately +4 °C in the dark.
Three slides per animal were incubated in alkaline (pH > 13) electrophoresis buffer to produce single-stranded DNA and to express ALS and SSB.
After alkali unwinding, electrophoresis was carried out at 1-10 °C, at 30 V (1 V/cm) and 300 mA, using a Bio-Rad power supply.
The times for unwinding specific to the tissue being investigated were: 1 hour for peripheral blood, 25 minutes for liver, 25 minutes for jejunum/ileum.
The times for electrophoresis specific to the tissue being investigated were: 35 minutes for peripheral blood, 25 minutes for liver, 25 minutes for jejunum/ileum.
Since not all slides can be processed at the same time, a block design was employed for the unwinding and electrophoretic steps in order to avoid excessive variation across the groups for each electrophoretic run.
After electrophoresis, the slides were immersed in 0.3 M sodium acetate in ethanol for approximately 30 minutes. Microgels were dehydrated in absolute ethanol for approximately 2 hours and immersed for 5 min in 70 % ethanol. Slides were air-dried and stored at room temperature. To avoid bias, the slides were randomly coded prior to scoring. Immediately before scoring, slides were stained with 12 µg/ml ethidium bromide.

Slides were examined at 400X magnification, with an image analysis system (Comet Assay II; Perceptive Instruments, UK) connected to a fluorescence microscope (Eclipse E400; Nikon).
Diffusion slides were scored by categorising 100 cells per slide into one of the two following groups:
- little or no diffusion from the nuclear “head”
- significant diffusion with either small or no head.
At least 50 morphologically normal cells were scored per slide/tissue to give a total number of 150 cells.
Hedgehog and cloud cells, if present, were excluded from the comet analysis.
Evaluation criteria:
DNA damage was evaluated as an extent of DNA migration. This extent of DNA migration is reflected in the tail intensity, which is generally defined as the fraction of DNA in the tail divided by the amount of DNA in the cell multiplied by 100, or the tail moment, which is the product of the tail length and the fraction of total DNA present in the tail.
For test item to be considered positive, a significant increase or decrease in DNA migration must be observed in any tissue evaluated, in at least one dose group. In addition there must be evidence of a dose-related increase or decrease in DNA migration measurements.
The experimental unit of exposure for in vivo studies is the animal and all analyses were based on individual animal response. The significance of the differences amongst groups was assessed by analysis of variance. Differences between each treated group and the control group were assessed by Dunnett's test using a pooled error variance (D.P. Lovell and T. Omori, 2008). The homogeneity of the data was verified by Bartlett's test before Dunnett's test. If data were found to be inhomogeneous a Modified t test (Cochran and Cox) was applied. The criteria for statistical significance were p<0.05, 0.01 and 0.001.

Results and discussion

Test results
Key result
no effects
Vehicle controls validity:
Negative controls validity:
see vehicle control
Positive controls validity:
Additional information on results:
- Dose range: 2000 and 1000 mg/kg bodyweight
- Clinical signs of toxicity in test animals: all animals showed blue spots on the back and on the muzzle one hour after dosing and along the duration of the study. In addition black spots in the litter were observed few hours after the first dosing and along the duration of the study. All animals showed piloerection one hour after the second dosing until sacrifice. Due to a possible misdosing, one female from the low dose group (1000 mg/kg) was found dead one hour after the second dosing.


Following treatment with the test item, blue faeces and blue spots were observed in the litter for all dose-levels. Blue spots on the back were observed in animals from the intermediate dose group and for one animal from the high dose group.
Blue spots on the tail were observed in all animals from the high dose group.
Following the second dosing, piloerection was observed in animals from the positive control group. These animals also showed slight body weight reductions.
At sacrifice all animals treated with the test item showed blue granular material in the stomach, intestine and a blue staining of the liver. No abnormality was observed in the bladder.

The number of “clouds” and the percentage of diffused cells were the parameters used to assess the number of cells with highly fragmented DNA. A high percentage of diffused cells combined with a high percentage of “clouds” on the comet slides indicates there may be excessive levels of cytotoxicity in the cell suspensions or the suspensions may not have been correctly prepared. Animals with > 30 % diffused cells may be excluded from data analysis in order to avoid any interference due to excessive cytotoxicity. A mild percentage of highly damaged cells was observed in cell suspensions prepared from jejunum/ileum of all animals with the exception of positive control group rats. However, based on the results obtained, no animal was excluded from scoring.

Treatment with E131 Patent blue induced a slight but statistically significant decrease (P<0.05) in tail moment over the vehicle control value in liver, only at the lowest dose level selected for treatment. This decrease was not confirmed when tail intensity was considered. A slight decrease over the vehicle control value was observed in peripheral blood at the intermediate dose level, when the parameter analysed was the tail intensity. This decrease was statistically significant (P<0.05) but was not confirmed by the analysis of tail moment. However, due to the absence of a dose-response relationship, to the magnitude of response and to the absence of a concurrent change in both measurements, these results were not considered biologically meaningful.
Marked increases in DNA migration were observed following treatment with the positive control item, indicating the correct functioning of the test system.

Any other information on results incl. tables


- Comet parameters of the vehicle control treatment are within historical control values and vehicle control data published in literature;

- The positive control treatment shows a statistically significant increase in Comet parameters over the concurrent vehicle control.

Applicant's summary and conclusion

Based on the results obtained from an in vivo alkaline comet assay, E131 Patent blue V sodium salt (CAS 20262-76-4) is not of concern for DNA damage.
Executive summary:

Patent blue V (E131) does not induce any effect in DNA migration in rat liver, jejunum/ileum and peripheral blood after in vivo treatment.