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Diss Factsheets

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:
From 2015-09-03 to 2015-10-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
no control of concentration in dosing formulations
Type of assay:
mammalian comet assay

Test material

Constituent 1
Chemical structure
Reference substance name:
[4-[p,p'-bis(dimethylamino)benzhydrylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium m-[[p-anilinophenyl]azo]benzenesulphonate
EC Number:
265-449-9
EC Name:
[4-[p,p'-bis(dimethylamino)benzhydrylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium m-[[p-anilinophenyl]azo]benzenesulphonate
Cas Number:
65113-55-5
Molecular formula:
C25H30N3.C18H14N3O3S
IUPAC Name:
[4-[p,p'-bis(dimethylamino)benzhydrylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium m-[[p-anilinophenyl]azo]benzenesulphonate
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River France, Saint-Germains-sur-l'Arbresle FRANCE
- Age at study initiation: 8 weeks old
- Weight at study initiation: between 185 g and 200 g
- Assigned to test groups randomly: yes, randpm-distribution
- Fasting period before study: A04C-10 from SAFE (batch 14294)
- Housing: bedding of dust-free, irradiated softwood pellets
- Diet (e.g. ad libitum): not fasted at the treatment time
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 8 days for the gentoxicity study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/-3 °C
- Humidity (%): 55 +/- 15°C with minor exceptions
- Photoperiod (hrs dark / hrs light): 12 hrs dark/12 hrs light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: non toxic vehicle which allows an homogenous solution/suspension.
- Amount of vehicle (if gavage or dermal):
- Lot/batch no. (if required): MKBP7039V for toxicity assays and MKB6944V for main assay
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
For the preliminary and confirmatory toxicity assays, suspensions at a maximum concentration of 200 mg/mL were prepared and administered to the animals at the dose volume of 10 mL/kg, giving a final dose of 2000 mg/kg. In the main genotoxicity assay, three suspensions of the test item at the concentrations of 200 - 100 and 50 mg/mL were prepared, giving final doses of 2000 - 1000 and 500 mg/kg, respectively when administered at 10 mL/kg.

Duration of treatment / exposure:
2 days
Frequency of treatment:
once per day
Post exposure period:
not applicable
Doses / concentrationsopen allclose all
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
0 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5 males per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
- Positive control: methylmethanesulfonate
- Justification for choice of positive control(s): positive control substance listed within the OECD 489. this positive control targets the 3 tissues evaluated within this study (liver, stomach, testis).
- Route of administration: oral (gavage)
- Doses / concentrations: 100 mg/kg bw/day

Examinations

Tissues and cell types examined:
Cells from liver, stomach and testis:
number of cells observed per animal: 150
number of cells observed per dose: 750.
Exminations performed on all aminals studied.

Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: One preliminary assay and one confirmatory assy were performed. The highest dose of 2000 mg/kg/day was the maximum tolerated dose, and in line with OECD 489 (2014).

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
2 treatments at 24-hrs intervals.
Sampling 2 to 6 hr after the last administration

DETAILS OF SLIDE PREPARATION:
Befor use, a volume of 85 µL of 0.8% of Normal agarose (NA) was added on the microscope slide prelayered with 1.5% of NA and then covered with a glass coverslip. Slides were placed at +2-8°C until the agarose layer hardened. The cells of the different doses tested were mixed with 0.5% of Low Melting Point Agarose (LMPA) (75 μL/slide) kept at ca. 37 °C and added on the microscope slide after gently sliding off the coverslip. The slides were then covered with a new glass coverslip, and were placed once again at +2-8°C.
Four slides per animal were prepared for the Comet assay.

METHOD OF ANALYSIS: Quantification of DNA damage
Evaluation criteria:
For a test item to be considered positive in the comet assay, it must be observed:
- At least one of the treatment groups exhibits a statistically significant increase in the mean of medians of percentage of DNA in tail compared with the concurrent negative control,
- This increase is dose-related at least at one sampling time when evaluated with an appropriate trend test, and
- Any of these results are outside the distribution of the historical negative control data.

When all of these criteria are met, the test chemical is then considered able to induce DNA strand breakage
in the tissues studied in this test system.


A test item is considered clearly negative if:
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- there is no concentration-related increase when evaluated with an appropriate trend test
- all results are inside the distribution of the historical negative control data for a given species, vehicle, route, tissue, and number of administrations
- direct or indirect evidence supportive of exposure of, or toxicity to, the target tissue(s) has been demonstrated.

The test chemical is then considered unable to induce DNA strand breakage in the tissues studied in this test system.
Statistics:
In order to quantify the test item effects on DNA, the following statistical analysis strategy was applied, using the statistical software Stat view®, version 5.
As the median of percentage of DNA in tail and other tail parameters do not follow a Gaussian distribution (E. Bauer et al., 1998), the non-parametric, one-way Kruskal-Wallis test was performed. This method is based on the analysis of variance by ranks for testing equality of population medians among groups.
The non-parametric Mann-Whitney U-test was applied to compare each of the doses tested with the vehicle control in order to determine statistical significance of differences in group median values between each group versus the vehicle control.

This test was also used to compare vehicle control and positive control to determine acceptable criteria of a valid test.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
no marked clinical signs. Noticed that the feaces were dark blue. No mortality occured.
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
Preliminary study
- Dose range: 2000 mg/kg bw/day (x2) per os, on 2 animals.
- Clinical signs of toxicity in test animals: slight sniffing after 15 min of both treatment and dark blue faeces 24 hour after the first treatment.
No mortality

Confirmatory study:
- Dose range: 2000 mg/kg bw/day -x2) per os, on 5 animals.
- Clinical signs of toxicity in test animals: no marked clinical signs and dark blue faeces 6 and 24 hours after the first treatment.
No mortality

Any other information on results incl. tables

Results in Liver Cells

GROUP

TEST ITEM

DOSES in mg/kg/day (2x)

% of DNA in tail. Mean of medians per animal (/5 animals*)

NON PARAMETRIC statistical assessment

Hedgehogs

p-Kruskall-Wallis

P Mann-Whitney

Relative ratio of hedgehogs

p

Negative control

Corn oil

0

0.36

N.S.

-

-

-

TREATED

Test item

2 000

0.27

N.S

1.15

N.S.

1 000

1.69

N.S.

1.24

N.S.

500

0.62

N.S.

1.35

N.S.

Positive control

Methylmethan Sulfonate

100

46.13

< 0.001

2.22

<0.05

Results in Stomach Cells

GROUP

TEST ITEM

DOSES in mg/kg/day (2x)

% of DNA in tail. Mean of medians per animal (/5 animals)

NON PARAMETRIC statistical assessment

Hedgehogs

p-Kruskall-Wallis

P Mann-Whitney

Relative ratio of hedgehogs

p

Negative control

Corn oil

0

16.54

N.S.

-

-

-

TREATED

Test item

2 000

7.91

< 0.05

1.06

N.S.

1 000

7.57

< 0.05

1.35

N.S.

500

7.36

< 0.05

1.00

N.S.

Positive control

Methylmethan Sulfonate

100

70.45

< 0.01

0.66

< 0.05

Results in Testis Cells

GROUP

TEST ITEM

DOSES in mg/kg/day (2x)

% of DNA in tail. Mean of medians per animal (/5 animals)

NON PARAMETRIC statistical assessment

Hedgehogs

p-Kruskall-Wallis

P Mann-Whitney

Relative ratio of hedgehogs

p

Negative control

Corn oil

0

2.58

N.S.

-

-

-

TREATED

Test item

2 000

1.05

< 0.05

0.94

N.S.

1 000

1.03

< 0.05

0.96

N.S.

500

1.27

N.S.

1.07

N.S.

Positive control

Methylmethan Sulfonate

100

38.26

< 0.001

0.59

<0.001

* for the high dose group in the liver analysis, only 4 animals were analysed

Applicant's summary and conclusion

Conclusions:
The test item induced no statistically significant increases in DNA strand breaks whatever the doses in male rat isolated Liver. Stomach and Testis cells after oral administration. Therefore, the test item is considered having no genotoxic activity in these organs.
Executive summary:

The genotoxic potential of the test item was investigated in the in vivo comet assay performed under alkaline conditions,i.e. pH > 13 (Alkaline Single Cell Gel Electrophoresis) in the male rat, in isolated Liver, Stomach and Testis cells, after two treatments at 24-hour interval by oral route (gavage) at 3 dose levels (2000, 1000 and 500 mg/kg), followed by one expression time of 2 to 6 hours after the last treatment.

A preliminary assay was performed on 2 male rats at the highest dose of 2000 mg/ kg/ day (x2) per os. It induced slight sniffing 15 minutes after both treatments.

It was noticed that, 24 hours after the first treatment, the feces were dark blue. No mortality was observed. These clinical signs were considered as acceptable and the dose of 2000 mg/ kg/ day (x2) per os was thus assessed for the induction of clinical signs during the confirmatory toxicity assay.

The confirmatory toxicity assay was performed on 5 male rats at the highest dose of 2000 mg/ kg/ day (x2) per os. It induced no marked climical signs. It was noticed that, 6 and 24 hrs after the first treatment, the feces were dark blue. No mortality was observed.

These clinical signs were considered as acceptable and the dose of 2000 mg/ kg/ day (x2) per os was thus retained for the comet assay. Two inferior doses of 1000 and 500 mg/kg/day (x2) per os were also tested.

The comet assay was performed on 5 males/group (4 analysed for the high dose group for liver), at 0 mg/kg b.w./day (vehicle alone - negative control), 500 mg/kg b.w./day, 1000 mg/kg b.w./day and 2000 mg/kg b.w./day.

5 additional aminals were treated orally twice with a positive control, the Methylmethane sulfonate, at a dose of 100 mg/kg

The rats were treated 2 times at 24 hours-intervals with the test item at the different doses. Two to six hours after the second treatment, the rats are euthanized and cells from the selected target organs (i.e. liver, stomach and testis) are isolated.

After isolation, single cells are embedded in agarose on microscope slides and the obtained microgels are successively submitted to lysis, unvinding and electrophoresis in alkaline conditions and under dimmedlight to prevent any additional DNa dmage. After neutralization, slides are dried and could therefore be stained with a fluorescent dye (e.g. propidium iodide) before analysis and scoring. The method used for quantifying DNA migration involves a computerized image analysis system in order to collect comet data; then, the dedicated software allows indeed the calculation of metrics for DNA migration.

Results in liver

No statistically significant increases in the mean of medians of percentage of DNA in tail were observed at the 3 tested doses of 2000, 1000 or 500 mg/kg/day (x2).

At the intermediary dose of 1000 mg/kg/day (x2), the mean of medians of percentage of DNA in tail was over the upper bound of historical data,i.e.1.69 vs.1.01. Nevertheless, this effect was due to heterogeneity between the 5 animals of this group. Indeed, one of the animals presented a median of percentage of DNA in tail higher than the other,i.e. 5.23vs. 0.19-1.58 while it was noticed that its liver presented an irregular appearance.

The test item was thus considered as not genotoxic toward the liver.

Results in stomach

No statistically significant increases in the mean of medians of percentage of DNA in tail were observed at the 3 tested doses of 2000, 1000 or 500 mg/kg/day (x2).

Statistically significant decreases in the percentage of DNA in tail were noted at all the tested doses without any dose-effect relationship. However, it has no meaning in terms of genotoxic hazard.

 

The test item was thus considered asnot genotoxic toward the stomach.

Results in testis

No statistically significant increases in the mean of medians of percentage of DNA in tail were observed at the 3 tested doses of 2000, 1000 or 500 mg/kg/day (x2).

Statistically significant decreases in the percentage of DNA in tail were noted at the two highest tested doses of 2000 and 1000 mg/kg/day (x2). However, it has no meaning in terms of genotoxic hazard.

 

The test item was thus considered as not genotoxic toward the testis.

CONCLUSION

The test item was investigated by the means of the in vivo comet assay under alkaline conditions (SCGE) in the Liver, Stomach and Testis of male rat treated orally twice with 2000, 1000 and 500 mg/kg/day, with one sampling time 2 to 6 hours after the last treatment according to OECD guideline (OECD 489, 2014).

 

The validity criteria for the study were met. The current study is thus considered as valid.

Under these experimental conditions, the test item induced no statistically significant increases in DNA strand breaks whatever the doses in male rat isolated Liver, Stomach and Testis cells after oral administration. Therefore, the test item is considered having no genotoxic activity in these organs.