Registration Dossier

Administrative data

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21.11. 2019 - 16.01.2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report Date:
2020

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Version / remarks:
adopted 29 July 2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EC method B.62 In Vivo Mammalian Alkaline Comet Assay, Commission Regulation (EU) No 2017/735
Version / remarks:
adopted 14 February 2017
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian comet assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Name of sample: VERSAL RED 2GC
Batch no. 157/19
Purity >98

Test animals

Species:
rat
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld , Germany
- Age at study initiation: 9 to 10 weeks
- Weight at study initiation:
M: 220 - 419 g
F: 203 - 262 g
- Assigned to test groups randomly: yes
- Fasting period before study: not specified
- Housing: The animals were kept in groups of 2 - 3 by sex in MAKROLON cages (type III plus). Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany) was used as bedding material for the cages. The cages were changed and cleaned twice a week.
- Diet: ad libitum
- Water: ad libitum
Certificates of analysis of diet, drinking water and bedding material are QAU archived.
- Acclimation period: 6 - 7 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 3°C
- Humidity (%): 55% ± 10%
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): The rooms were lit (about 150 lux at approx. 1.50 m room height) and darkened for periods of 12 hours each.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: 0.8% aqueous hydroxypropylmethylcellulose - Methocel, batch no. 13D03-N03, Fagron GmbH & Co. KG, 22885 Barsbüttel, Germany

- Justification for choice of solvent/vehicle: 0.8% aqueous hydroxypropylmethylcellulose was chosen as vehicle as it is known not to produce any toxic effects according to known available information.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

VERSAL RED 2GC was suspended in 0.8% aqueous hydroxypropylmethylcellulose. The administration volume was 20 mL/kg b.w..

In line with normal practice in this type of in vivo study, no analysis of the dose form is required. A test on stability and homogeneity was not necessary as the test item formulation was prepared immediately before administration.


Duration of treatment / exposure:
45 hours
Frequency of treatment:
Three times at 0, 24 and 45 hours
Post exposure period:
3 hours
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
vehicle control
Dose / conc.:
400 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5 animals
Control animals:
yes, concurrent vehicle
Positive control(s):
ethylmethanesulphonate
- Justification for choice of positive control(s): not specified, it is an example of positive control substance from OECD 489 for any tissue
- Route of administration: oral by gavage, administered two times at 24 and 45 hours
- Doses / concentrations: 125 mg/kg b.w./day

Examinations

Tissues and cell types examined:
M: liver, glandural stomach, duodenum cells, testicle
F: ovaries
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
The dose levels were selected based on the results of a Limit preliminary experiment to determine the maximum-tolerated-dose (MTD) using two animals per dose. Based on the results the maximum-tolerated dose level (100%; 2000 mg/kg b.w./day, p.o), a medium-tolerated dose level (50% MTD) and a lower dose (20% MTD) were tested. No signs of systemic toxicity were noted at the highest dose level of 2000 mg/kg b.w./day, p.o.
In line with normal practice in this type of in vivo study, no analysis of the dose formulation is required.


TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Animals were treated with the test item at three intervals of 0, 24 and 45 hours and liver tissues were sampled at 48 hours (3 hours after the last dose) in order to accommodate the sampling requirements of the comet assay. Treatment with the positive reference item was performed twice: at 24 and 45 hours (3 hours before sampling).

DETAILS OF SLIDE PREPARATION:
For the preparation of slides the CometAssay® HT Kit (Trevigen) was used according to the manufacturer´s instructions. Each cell suspension was mixed 1:10 with low-melting agarose. An adequate amount of the cell-agarose mixture was applied to two wells of the 20-well microscope slide. The preparation of the slides was completed within two (2) hours after sacrifice.
The prepared slides were incubated in a lysis buffer overnight at +2°C to +8°C. After incubation, the slides were incubated in freshly prepared alkaline unwinding solution (pH>13) for 20 minutes at room temperature.

METHOD OF ANALYSIS:
The slides were analysed by epifluorescence microscopy at 200 x magnification. At least 150 liver cells per animal and tissue were evaluated using the Comet Assay IV software (Perceptive Instruments Ltd).
Evaluation criteria:
Percent tail DNA (% tail intensity) was determined to assess DNA damage. The DNA fragment intensity in the tail was expressed as a percentage of the cell's total intensity. The median % tail DNA for each animal was calculated. The mean of the individual animal’s median was then determined.

Only cells of good morphology (clearly defined head and tail with no interference with neighbouring cells) were scored for % tail DNA. Artefacts were avoided. The occurrence of hedgehogs was determined based on the visual scoring and tabulated but not evaluated and reported.
Statistics:
Generally, means and standard deviations were calculated. Intergroup comparisons with the control group were made by an analysis of variance (ANOVA) followed by the DUNNETT multiple comparison test (p ≤ 0.05 and p ≤ 0.01) for the cells of liver and testis. A square root transformation of the data was needed in the case of stomach cells because of the high standard deviation. In addition, a nonparametric ANOVA test (Kruskal-Wallis Test) was needed in the case of duodenum and ovary cells, due to the failing of the normality test. The positive control data were compared to the values of the vehicle control group values by unpaired Student's t-test (t) (normal distribution) (WILCOXON MANN-WHITNEY Test with StatXact 4 (not normal distributed). Significantly different data were indicated in the tables of the report. A possible dose-response-relationship would have been examined by linear regression analysis employing PEARSON's correlation coefficient.

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
In conclusion, under the present test conditions VERSAL RED 2GC tested up to the highest reasonable dose level of 2000 mg/kg b.w. per day by repeated oral administration to male and female rats for three days showed no genotoxic properties in the in vivo alkaline comet (single cell gel electrophoresis) assay employing cells of the liver, glandular stomach, duodenum and testis (males) or ovaries (females).
In the same system, ethyl methane sulfonate (positive reference item) induced significant damage.
Executive summary:

VERSAL RED 2GC was assayed in an in vivo alkaline comet (single cell gel electrophoresis) assay for the detection of DNA strand breaks in cells or nuclei isolated from liver, glandular stomach, duodenum and testis tissue of male CD rats, as well as ovary tissue from female CD rats. The test item was administered orally to rats once daily for three days at 0, 24 and 45 hours, and the tissues were sampled at 48 hours (3 hours after the last dose).

VERSAL RED 2GC was suspended in 0.8% aqueous hydroxypropylmethylcellulose. The dose levels for the main study had been selected based on the result of a preliminary Limit test of 2000 mg/kg b.w./day, p.o employing two animals per dose. No signs of systemic toxicity were noted.

Three ascending dose levels of 400, 1000 or 2000 mg VERSAL RED 2GC/kg b.w./day, p.o. and the vehicle (0.8% aqueous hydroxypropylmethylcellulose) were administered three times at 0, 24 and 45 hours. The positive reference item (125 mg ethyl methane sulfonate/kg b.w./day, p.o.) was administered two times at 24 and 45 hours. Each group consisted of 5 male and 5 female rats. The administration volume was 20 mL/kg b.w./day. The test was divided in 4 different preparation groups containing one or two animals of each group which were tested in parallel at each stage of the experiment.

No signs of systemic toxicity were noted up to the highest dose level of 2000 mg VERSAL RED 2GC/kg b.w./day, p.o.

150 cells of liver, glandular stomach, duodenum and testis (males) or ovaries (females) per animal and tissue collected 48 hours post 1st administration were evaluated. The grade of DNA fragmentation is expressed in percent tail DNA (% tail intensity).

Treatment with VERSAL RED 2GC did not cause any test item-related increase in the tail intensity for the cells of liver, glandular stomach, duodenum and testis (all males) or ovaries (females) at any of the three tested dose levels of 400, 1000 and 2000 mg test item/kg b.w./day compared to the vehicle control data.

The group mean values of the median tail intensities of the test item treatment groups were between 6.325% and 7.83%, 9.58% and 11.806% or 11.554% and 12.856% for liver, stomach and duodenum cells, respectively. The corresponding values for vehicle control were 4.726%, 8.152% or 12.554% for liver, stomach and duodenum cells, respectively. The mean values of the median tail intensity were between 5.341% and 6.220% or 1.082% and 1.681% for the cells of testis and ovaries, respectively. The corresponding values for vehicle control were 5.326% or 1.782% for the cells of testis and ovaries, respectively.

All values obtained were within the upper limits of the historical control data, except for the vehicle control group of stomach cells and test item treatment groups of liver and stomach cells which were slightly above the background data. As the slightly higher data are no extreme outliers, were within published historical control data and older rats at an age of 9 – 12 weeks were used, to assure maternity of the animals for evaluation of testis and ovary cells, the results are considered acceptable for the inclusion into the historical control data and the test is considered valid.

Administration of ethyl methane sulfonate (positive reference) significantly increased the % tail intensity for the cells of liver, glandular stomach, duodenum and testis (all males) or ovaries (females).

All values were well within the historical background data, except the positive control for the stomach and duodenum cells which were above the historical background data. As the positive control is significantly increased, the data are considered acceptable for the inclusion into the historical control data and the test is considered valid.

 

In conclusion, under the present test conditions VERSAL RED 2GC tested up to the highest reasonable dose level of 2000 mg/kg b.w. per day by repeated oral administration to male and female rats for three days showed no genotoxic properties in the in vivo alkaline comet (single cell gel electrophoresis) assay employing cells of the liver, glandular stomach, duodenum and testis (males) or ovaries (females).

In the same system, ethyl methane sulfonate (positive reference item) induced significant damage.