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Genetic toxicity in vitro

Description of key information

The test substance Semi Dry Absorption (SDA) Product was examined for its mutagenicity potential by the following tests:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

All of the above mentioned tests were carried out in accordance with GLP principles.

Under the described experimental design, the test substance Semi Dry Absorption (SDA) Product was non-mutagenic for all the Salmonella typhimurium as well as Escherichia coli strains both in experiments without as well as with metabolic activation.

The test substance, Semi Dry Absorption (SDA) Product, did not show any genotoxic effect with or without metabolic activation in the chromosome aberration test under given experimental condition. Negative effect in in vitro chromosome aberration test indicates that the test substance did not induce chromosome aberrations in culture of human somatic cells.

The test item Semi Dry Absorption (SDA) Product is considered to be mutagenic in the mouse lymphoma thymidine kinase locus using the cell line L5178Y with metabolic activation.

Endpoint Conclusion: No adverse effect observed (negative)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31.07. - 21.09.2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
yes
Remarks:
without impact (see Overall remarks)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
gene for synthesis histidine or tryptophan
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: histidine requiring strain
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
other: tryptophan requiring strain
Metabolic activation:
with and without
Metabolic activation system:
post-mitochondrial fraction (S9)
Test concentrations with justification for top dose:
5, 15, 50, 150, 500 μg/plate
For justification for top dose see box Additional information on results.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water for injection, Ardeapharma a.s., Lot No. 0101030309
- Justification for choice of solvent/vehicle: solubility of the test substance
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylenediamine
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 9-aminoacridine hydrochloride monohydrate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminofluorene
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-methyl-N'-nitro-N-nitrosoguanidine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
100 ul of Semi Dry Absorption (SDA) Product suspension of required concentration, 0.1 ml 16-18h culture of the tester strain, 0.5 ml relevant buffer and 30 or 100 ul of S9 postmitochondrial fraction (in case of test with metabolic activation) were added to the 2 ml top agar (with trace of histidine or tryptophan) kept in a test tube at 45± 3°C.
After shaking the mixture was poured into a minimal glucose agar plate.

The number of revertant colonies on the plate was counted manually or by using an AccuCount 1000 after 48 - 72 h incubation at 37± 1°C.

NUMBER OF REPLICATIONS: 2 series; each with and without metabolic activation, with positive control and solvent control, triplicate plating was used at each dose level

DETERMINATION OF CYTOTOXICITY
- Method: total growth
Evaluation criteria:
The main criterion for evaluation of results was modified two-fold increase rule, its using is comparable with using of statistical methods (2, 3). After this rule the result is positive, when reproducible dose-effect and/or doubling of ratio Rt/Rc is reached.
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
COMPARISON WITH HISTORICAL CONTROL DATA:
Spontaneous reversions, negative controls (solvent), and positive controls were compared with historical controls in our laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
As the test substance is almost insoluble in any solvent, a suspension was prepared in maximum concentration given in guidelines (5000 µg per plate). This concentration was further diluted and the concentration row arised (10-5000 µg per plate) was tested for toxicity in strain TA 100 without metabolic activation. The test substance increased number of revertants gradually – none of them was toxic according to rules given in SOP M/12/10. Bacterial background was hardly evaluable because of particles of the test substance but it seemed normal.
Due to the decrease of number of revertants the first mutagenicity test in TA 100 without metabolic activation was done with higher number of doses than usually. Slow decrease was confirmed and the dose of 1500 µg per plate was toxic. The highest non- toxic dose (but dose with decreased number of revertants) -500 µg per plate- was used as maximum for the first mutagenicity experiments. This dose was diluted according to the rules given in guidelines for origination of concentration row.
No dificulties in evaluation showed at any dose, therefore the second mutagenicity experiments were done with the same doses.
All doses were dosed in amount of 0.1mL. Fresh suspensions of test substance were prepared before each experiment. The suspensions were shaken at dilution, before dosing to top agar and before pouring onto plates.
Conclusions:
Under the above-described experimental design, the test substance Semi Dry Absorption (SDA) Product was non-mutagenic for all the Salmonella typhimurium as well as Escherichia coli strains both in experiments without as well as with metabolic activation.
Executive summary:

Test substance Semi Dry Absorption (SDA) Product was assayed for the mutagenicity by the Bacterial Reverse Mutation Test. The test was performed according to EU method B.13/14 Mutagenicity – Reverse mutation test using bacteria, which is analogous to the OECD Test Guideline No. 471.

Four indicator Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 and one indicator Escherichia coli WP2 uvrA strain were used. The test substance was suspended in water for injection and assayed in doses of 5-500 ug which were applied to plates in volume of 0.1 mL.

Two series of experiments were performed with each strain - without metabolic activation and with a supernatant of rat liver and a mixture of cofactors.

In the arrangement given above, the test substance Semi Dry Absorption (SDA) Product was non-mutagenic for all the used bacterial strains with as well as without metabolic activation.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05.02. - 28.04.2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
2000
Deviations:
yes
Remarks:
(see Overall remarks part)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: Human lymphocytes obtained by cell culture of whole blood from peripheral blood of healthy volunteers.
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640, Sevapharma, Fetal bovine serum, Gibco
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction of rat liver homogenate (acquired commercially) and a mixture of cofactors
Test concentrations with justification for top dose:
Test substance was extracted in cultivation medium at a concentration 1 g/5 ml for 72 hours. Final dilutions used for testing were 2 mg/ml (100x diluted), 200 mg/ml (1000x diluted) and 20 mg/ml (10,000x diluted).
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: RPMI 1640, Sevapharma, Fetal bovine serum, Gibco
Untreated negative controls:
yes
Remarks:
untreated culture, metabolic activation system
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
without metabolic activation
Untreated negative controls:
yes
Remarks:
untreated culture, metabolic activation system
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation
Untreated negative controls:
yes
Remarks:
untreated culture, metabolic activation system
Positive controls:
yes
Positive control substance:
other: thiotepa
Remarks:
without metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 72 h
- Exposure duration: 24 h
- Expression time (cells in growth medium): 20 h

SPINDLE INHIBITOR: Colchicine
STAIN: Giemsa dye

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 200

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity was evaluated using the WST-1 and neutral red tests.
Evaluation criteria:
Genotoxic effect of the test substance was evaluated as a percentage of aberrant metaphases; i.e. cells in metaphase positive for the below-mentioned types of damage against the total number of evaluated metaphases. In accordance with internal SOP M/42/3, aberrations of the type gap were not included in the total count of mutagenic effects.

Criteria of acceptance / Interpretation of results
a) the percentage of aberrant metaphases in controls did not exceed 3 %
b) genotoxic effect of the test substance is manifested in case of a number of aberrant metaphases being higher than 5 %
Species / strain:
lymphocytes: Human lymphocytes obtained by cell culture of whole blood from peripheral blood of healthy volunteers.
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
only in prelimary test yes - in the highest dose (see part Any other information on results)
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested

Preliminary experiments

Significant cytotoxicity was not found except the highest extract concentration of the test substance (2 mg SDA /ml). Thus for the purpose of genotoxicity testing, three highest SDA concentrations were selected.

Test substance has been extracted in cultivation medium at a concentration 1 g/5 ml which is the maximal concentration of chemical to be tested as recommended by the particular guidelines. This dose represented maximal concentration and did not show any toxicity in preliminary tests (viability-cytotoxicity, osmolarity and pH).

Conclusions:
Result: negative with/without metabolic activation
The test substance, Semi Dry Absorption (SDA) Product, did not show any genotoxic effect with or without metabolic activation in the above-mentioned chromosome aberration test under given experimental condition. Negative effect in in vitro chromosome aberration test indicates that the test substance did not induce chromosome aberrations in culture of human somatic cells.
Executive summary:

Test substance, Semi Dry (SDA) Absorption Product, was subjected to in vitro chromosome aberration test according to the EU method B10. Mutagenicity - In vitro mammalian chromosome aberration test, Directive 2000/32/EC, published in OJ L 136, 2000.

Human lymphocytes obtained from peripheral blood of healthy volunteers were used for the purpose of test. Test substance was extracted in cultivation medium at a concentration 1 g/5 ml for 72 hours. Final dilutions used for testing were 2 mg/ml (100x diluted), 200 microg/ml (1000x diluted) and 20 microg/ml (10,000x diluted).

Experiments were carried out both with and without metabolic activation using supernatant of hepatic homogenate of rats (S9 fraction) and a mixture of cofactors.

Using the above-mentioned arrangement of test, the test substance, Semi Dry Absorption (SDA) Product – did not show any genotoxic effect in all experiments with or without metabolic activation. Negative effect in in vitro chromosome aberration test indicates that the test substance did not induce chromosome aberrations in the cultures of human somatic cells.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24.02. - 29.03.2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
May 2008
Deviations:
yes
Remarks:
(reason: typing error; deviation did not influence the quality or integrity of study)
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine Kinase Locus/TK+/-
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 complete medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
S9 liver microsomal fraction with cofactor
Test concentrations with justification for top dose:
Experiment I
with metabolic activation: 1000, 2000, 2500, 2800, 3600, 4000, 4500 and 5000 μg/mL
and without metabolic activation: 60, 125, 250, 500, 1000, 1200, 1350 and 1500 μg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: RPMI (Roswell Park Memorial Institute medium) + 3% HS (Horse Serum) for short-term exposure, RPMI + 7.5% HS for long-term exposure
Untreated negative controls:
yes
Remarks:
(RPMI medium supplemented with HS)
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation
Untreated negative controls:
yes
Remarks:
(RPMI medium supplemented with HS)
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without metabolic activation
Untreated negative controls:
yes
Remarks:
(RPMI medium supplemented with HS)
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h (short-term exposure), 24 h (long-term exposure)
- Expression time (cells in growth medium): 72 h (short-term exposure), 48 h (long-term exposure)
- Selection time (if incubation with a selection agent): 11-14 days

SELECTION AGENT (mutation assays): RPMI 1640 complete culture medium supplemented with TFT

NUMBER OF REPLICATIONS: experiment I was performed with and without metabolic activation

DETERMINATION OF CYTOTOXICITY
- Method: total growth
Evaluation criteria:
- the mutant frequency
- the colony sizing
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
(4000 and 5000 µg/mL with metabolic activation)
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(for the highest concentration 5000 µg/mL in experiment I with metabolic activation and 1500 µg/mL without metabolic activation)
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test item was noted in experiment I at concentrations of 1000 µg/mL and higher ( with and without metabolic activation)

RANGE-FINDING/SCREENING STUDIES: The selection of the concentrations used in the main experiments was based on data from pre-experiments according to the OECD guideline 476.

COMPARISON WITH HISTORICAL CONTROL DATA:
In experiment I with metabolic activation all mutant values found were within the historical control data of the test facility.
Without metabolic activation all mutant values found were within the historical control data of the test facility.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Growth inhibition was observed in experiment I with and without metabolic activation. In experiment I with metabolic activation the relative total growth (RTG) was 14.96% for the highest concentration (5000 µg/mL) evaluated. The highest concentration evaluated without metabolic activation was 1500 µg/mL with RTG of 14.49%.
Remarks on result:
other: all strains/cell types tested
Conclusions:
Interpretation of results:
positive with metabolic activation
negative without metabolic activation

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item Semi Dry Absorption (SDA) Product is considered to be mutagenic in the mouse lymphoma thymidine kinase locus using the cell line L5178Y with metabolic activation.
Executive summary:

The test item Semi Dry Absorption (SDA) Product was assessed for its potential to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

The selection of the concentrations was based on data from the pre-experiments. In experiment I 5000 μg/mL ( with metabolic activation) and 1500 μg/mL ( without metabolic activation) were selected as the highest concentrations.

The test item was investigated at the following concentrations:

Experiment I

with metabolic activation:

1000, 2000, 2500, 2800, 3600, 4000, 4500 and 5000 μg/mL

and without metabolic activation:

60, 125, 250, 500, 1000, 1200, 1350 and 1500 μg/mL

Precipitation of the test item was noted with and without metabolic activation at concentrations of 1000 μg/mL and higher.

Growth inhibition was observed in experiment I with and without metabolic activation.

In experiment I with metabolic activation the relative total growth (RTG) was 14.96% for the highest concentration (5000 μg/mL) evaluated. The highest concentration evaluated without metabolic activation was 1500 μg/mL with a RTG of 14.49%.

In experiment I a biologically relevant increase of mutants was found after treatment with the test item ( with metabolic activation). A dose-response relationship was observed.

Additionally, in experiment I colony sizing showed clastogenic effects induced by the test item under the experimental conditions ( with metabolic activation).

EMS, MMS and B[a]P were used as positive controls and showed distinct and biologically relevant effects in mutation frequency. Additionally, MMS and B[a]P significantly increased the number of small colonies, thus proving the efficiency of the test system to indicate potential clastogenic effects.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

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.

Link to relevant study records
Reference
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
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
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.
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
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
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.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid

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.

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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

Both bacterial reverse mutation test and chromosome aberration assay led to negative results. Result of the mammalian cell gene mutation assay was inconclusive. Futher the test substance showed no genotoxic properties in the in vivo alkaline comet (single cell gel electrophoresis) assay employing liver, stomach and duodenum cells therefore the conclusion is negative in relation to genetic toxicity.