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Toxicological information

Genetic toxicity: in vivo

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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:
26.06. - 07.09.2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Version / remarks:
adopted 29 July 2016
Deviations:
yes
Remarks:
See Any other information
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 test material (as cited in study report): Product of Semi-Dry Absorption method of Flue Gas Desulphurization (SDA Product)
- Molecular formula (if other than submission substance): not available
- Molecular weight (if other than submission substance): not available

Composition of test substance
CaSO4*H2O 27.46 %
CaCO3 10.48 %
CaSO3*1/2H2O 34.83 %
CaCl2*2H2O 15.30 %
CaF2 0.62 %
Ca(OH)2 7.78 %
CaO free 5.89 %
Loss by ignition 10.67 %
Sum of toxic metals
(As, Ba, Cd, Co, Cr, Cu, Hg, Mo, Ni, Pb, Sb, Se, Te, Ti, V, Zn)….< 0.1 %

Batch No.: SDA 13.12.16 SKW
Appearance: light grey solid powder

Test animals

Species:
rat
Strain:
Crj: CD(SD)
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: 7 to 9 weeks
- Weight at study initiation: 210 - 300 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: 7 days

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:
Product of Semi-Dry Absorption method of Flue Gas Desulphurization (SDA Product) was suspended in 0.8% aqueous hydroxypropylmethylcellulose. The administration volume was 20 mL/kg b.w.
Duration of treatment / exposure:
45
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: 250 mg/kg b.w./day

Examinations

Tissues and cell types examined:
liver, stomach and duodenum cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
The dose levels for the main study had been selected based on the results of a preliminary dose-range-finding study of 250, 500, 1000 and 2000 mg/kg b.w./day, p.o. employing two animals per dose. No signs of systemic toxicity were noted up to the highest dose level of 2000 mg/kg b.w./day, p.o.
All groups were used for the sampling. The highest dose for the comet assay is defined as the dose producing signs of toxicity such that higher dose levels, based on the same dosing regimen, would be expected to produce lethality.
As no toxicity occurred the highest reasonable dose level of 2000 mg/kg b.w. was employed.
In line with normal practice in this type of in vivo study, no analysis of the dose form 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 tissues (liver, stomach and duodenum) 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: 0 and 21 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, stomach and duodenum cells per animal and tissue were evaluated using the Comet Assay IV software (Perceptive Instruments Ltd).
All slides for analysis, including those of positive and negative controls were independently coded.
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 test7 (p ≤ 0.05 and p ≤ 0.01). A square root transformation of the data was needed because of the high standard deviation. The positive control data were compared to the values of the vehicle control group values by unpaired Student's t-test (t)8 (normal distribution) (WILCOXON MANN-WHITNEY Test with StatXact 49 (not normal distributed). Significantly different data were indicated in the tables of the report. A possible dose-response-relationship was examined by linear regression analysis employing PEARSON's correlation coefficient.

Results and discussion

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

Any other information on results incl. tables

Deviation:

The study was conducted in accordance with the Study Plan agreed upon. There were no major deviations from the Study Plan. However, the following minor deviations were noted:

• The sampling time of the positive reference item was 24-hours and not 48-hours as stated in the Study Plan. This was caused by copy/paste.

• The animal numbers of the 5 tested groups differ from Study Plan. The test was separated in 2 different preparations groups containing 2 or 3 animals of each group, respectively: animal nos. 1 to 10 and animal nos. 11 to 25 were tested each as one parallel experiment.

These minor deviations from the Study Plan did not affect the scientific outcome or the validity of the study.

Applicant's summary and conclusion

Conclusions:
In conclusion, under the present test conditions Product of Semi-Dry Absorption method of Flue Gas Desulphurization (SDA Product) tested up to the highest reasonable dose level of 2000 mg/kg b.w. per day by repeated oral administration for three days showed no genotoxic properties in the in vivo alkaline comet (single cell gel electrophoresis) assay employing liver, stomach and duodenum cells. In the same system, ethyl methane sulfonate (positive reference item) induced significant damage.
Executive summary:

Product of Semi-Dry Absorption method of Flue Gas Desulphurization (SDA Product) 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, stomach and duodenum, tissues of male CD rats. The test item was administered orally to rats once daily for three days at 0, 24 and 45 hours, and liver, stomach and duodenum tissues were sampled at 48 hours (3 hours after the last dose).

Product of Semi-Dry Absorption method of Flue Gas Desulphurization (SDA Product) was suspended in 0.8% aqueous hydroxypropylmethylcellulose. The dose levels for the main study had been selected based on the results of a preliminary dose-range-finding study of 250, 500, 1000 and 2000 mg/kg b.w./day, p.o employing two animals per dose. No signs of systemic toxicity were noted up to the highest dose level of 2000 mg/kg b.w./day, p.o.

Three ascending dose levels of 400, 1000 or 2000 mg Product of Semi-Dry Absorption method of Flue Gas Desulphurization (SDA Product)/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 (250 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 rats. The administration volume was 20 mL/kg b.w./day.

No signs of systemic toxicity were noted up to the highest dose level of 2000 mg Product of Semi-Dry Absorption method of Flue Gas Desulphurization (SDA Product)/kg b.w./day, p.o.

150 liver, stomach and duodenum cells 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 Product of Semi-Dry Absorption method of Flue Gas Desulphurization (SDA Product) did not increase the tail intensity for the liver, stomach and duodenum cells at any of the three tested dose levels of 400, 1000 or 2000 mg test item/kg b.w./day compared to the vehicle control data. The vehicle results were within the historical control ranges. Administration of ethyl methane sulfonate (positive reference) significantly increased the % tail intensity of liver, stomach and duodenum cells. Therefore, the test is considered valid.

 

In conclusion, under the present test conditions Product of Semi-Dry Absorption method of Flue Gas Desulphurization (SDA Product) tested up to the highest reasonable dose level of 2000 mg/kg b.w. per day by repeated oral administration for three days showed no genotoxic properties in the in vivo alkaline comet (single cell gel electrophoresis) assay employing liver, stomach and duodenum cells.

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