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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Bacterial reverse mutation assay

In a K1 well documented and GLP compliant bacterial reverse mutation assay in Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and in the Escherichia coli strain WP2uvrA, performed according to OECD Guideline 471, it was concluded that 4nitrobenzene-1 -sulfonyl chloride has no mutagenic effects on the strains tested in absence and presence of S9 -mix under the test conditions described in the report.

- In vitro mammalian cell micronucleus test

In a K1 well documented and GLP compliant in vitro mammalian cell micronucleus test with cultured peripheral human lymphocytes, performed according to OECD 487, it is concluded that the test item is not clastgenic or aneugenic in human lymphycotes under the experimental conditions described in the report.

- Gene mutation study in mammalian cells

In a K1 well-documented and GLP compliant mouse lymphoma assay, it was concluded that the test item is mutagenic in the mouse lymphoma L5178Y test system in the presence of S9-mix at toxic dose levels. The test item is not mutagenic in the mouse lymphoma L5178Y test system in the absence of S9-mix.

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:
2017-08-10 to 2018-03-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: M16LB4523
- Expiration date of the lot/batch: 2018-06-23
- Purity test date: 2018-04-17

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: not indicated
- Solubility and stability of the test substance in the solvent/vehicle: not indicated

OTHER SPECIFICS: A correction factor of 1.00 for the purity/composition of the test item was applied in this study.
Target gene:
Histidine locus (histidine-dependent S. typhimurium strains); Tryptophan locus (tryptophan-dependent E. coli strains)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (Aroclor 1254 induced rat liver metabolic activation system)
Test concentrations with justification for top dose:
The maximum final concentration for the dose range finding test was based on thesolubility of the test item in DMSO (maximum recommended dose = 5000 µg/plate).
Dose-range finding test: 1.7, 5.4, 17, 164, 512, 1600 and 5000 μg/plate in TA100 and WP2uvrA with and without 5% (v/v) S9-mix

The top doses for the mutation experiments were selected based on the toxicity observed in the dose range finding test.
Mutation experiment 1: 17, 52, 164, 512, 1600 and and 5000 μg/plate in TA1535, TA1537 and TA98 with and without 5% (v/v) S9-mix
Mutation experiment 2: 188, 375, 750, 1250, 2500 and and 5000 μg/plate with and without 10% (v/v) S9- mix

Mutation experiment 3: To verify the mutagenic response observed in tester strain TA98 with and without 10% (v/v) S9-mix, this part of the experiment was repeated with the following doses: 1000, 1500, 2000, 2500, 3000, 3500 and 4000 μg/plate

Since the test item is not stable in DMSO, the mutation experiments were repeated with anhydrous THF as solvent in additional fourth and fifth mutation assays.
The maximum final concentration was based on the stability and solubility of the test item in THF (maximum recommended dose = 5000 µg/plate) and toxicity observed in previous experiments.
Mutation experiment 4: 17, 52, 164, 512, 1600 and 5000 μg/plate; with and without 5% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA.

Mutation experiment 5: 5.12, 12.8, 32, 80, 200, 500 and 750 μg/plate; with and without 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA.

Mutation experiment 6: To verify the mutagenic response observed in the tester strains TA98 and TA1537 in the fifth mutation experiment, this part of the experiment was repeated.
without S9-mix in tester strain TA98: 9.4, 18.8, 37.6, 75, 100, 150 and 200 μg/plate,
with 5% (v/v) S9-mix in tester strain TA1537: 5.12, 12.8, 32, 80, 200 and 500 μg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: anhydrous tetra hydrofuran (THF)
- Justification for choice of solvent/vehicle: The test item was observed to be insoluble in water at 50 mg/ml. In DMSO, the test item was soluble at 50 mg/ml (= 5000 μg/plate). Based on these solubility findings, DMSO was selected as vehicle and 5000 μg/plate was selected as the maximum final concentration for the dose range finding test. However, due to the instability of the test item in DMSO, the mutation experiments were repeated with anhydrous tetra hydrofuran (THF) as vehicle. The test item was soluble at 200 mg/ml in THF and 5000 μg/plate was selected as the maximum final concentration for the fourth mutation experiment.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO / THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Without S9-mix; 5 μg/plate (TA1535)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO / THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: OCR-191
Remarks:
Without S9-mix; 2.5 μg/plate (TA1537)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO / THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Without S9-mix; 10 μg/plate (TA98)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO / THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Without S9-mix; 650 μg/plate (TA100)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO / THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Without S9-mix; 10 μg/plate (WP2uvrA)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO / THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
2.5 μg/plate (TA1535 with 5 and 10% S9-mix, TA1537 with 5% S9-mix), 5 μg/plate (TA1537 with 10% S9-mix), 1μg/plate (TA98 with 5 and 10% S9-mix, TA100 with 5% S9-mix), 2 μg/plate (TA100 with 10% S9-mix), 15 μg/ plate (WP2uvrA with 5 and 10% S9-mix)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
Top agar in top agar tubes was melted by heating to 45 ± 2°C.
The following solutions were successively added to 3 ml molten top agar:
- 0.1 ml of a fresh bacterial culture (10^9 cells/ml) of one of the tester strains
- 0.1 or 0.025 ml of a dilution of the test item in DMSO or THF, respectively and
- either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of nonactivation assays).
The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies were counted.

DURATION
- Exposure duration: 48 ± 4 h
- Selection time (if incubation with a selection agent): 48h (simultaneous with exposure)

SELECTION AGENT (mutation assays): Histidine (S. typhimurium histidine-dependent strains); Tryptophan (E. coli tryptophan-dependent strains)

NUMBER OF REPLICATIONS: all concentrations for all experiments were tested in triplicate

DETERMINATION OF CYTOTOXICITY
- Method: Reduction in bacterial background lawn; increase in size of the microcolonies; reduction of revertant colonies
Evaluation criteria:
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent vehicle control, or the total number of revertants in tester strain TA1535, TA1537 or TA98 is greater than three (3) times the concurrent vehicle control.
b) A concentration related effect is observed.
c) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

In addition to the criteria stated above, any increase in the total number of revertants was evaluated for its biological relevance including a comparison of the results with the historical control data range.
Statistics:
No formal hypothesis testing was done.
Species / strain:
S. typhimurium, other: TA1535, TA1537, TA98 and TA100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test item was observed to be insoluble in water at 50 mg/ml.
- Precipitation:
Mutation experiment 1, 2 and 3: Precipitation of the test item on the plates was not observed at the start or at the end of the incubation period in any tester strain.
Mutation experiment 4: Precipitation of the test item on the plates was observed at the start of the incubation period at concentrations of 512 μg/plate and upwards and at 164 μg/plate and above at the end of the incubation period in all tester strains in the absence and presence of S9-mix.
Mutation experiment 5: Precipitation of the test item on the plates was observed at the start of the incubation period at concentrations of 500 μg/plate and upwards and at 80 μg/plate and above at the end of the incubation period in all tester strains in the absence and presence of S9-mix
Mutation experiment 6: Precipitation of the test item on the plates was observed at the start of the incubation period at concentrations of 500 μg/plate and at 200 μg/plate and above at the end of the incubation period in both tester strains.

RANGE-FINDING/SCREENING STUDIES:In the dose range finding test, the test item was tested at concentrations of 1.7, 5.4, 17, 164, 512, 1600 and 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA100 and WP2uvrA. It was reported as part of the first mutation experiment.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
The vehicle control values were within the laboratory historical control data ranges, except the response for TA1537 in the fifth experiment (presence of S9-mix). Since the mean number of revertant colonies showed a slightly lower number of revertant colonies (2 revertant colonies) when compared against relevant historical control data (3 revertant colonies), the validity of the test was considered not to be affected. In addition, this tester strain was repeated in the sixth experiment.
The strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly, except the response for TA1535 (fifth experiment, absence of S9-mix). The positive control is included as a reference for the test system, where a positive response is required to check if the test system functions correctly. Since the value was above the historical control data range, this deviation in the mean plate count of the positive control had no effect on the validity of the results.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Mutation Expeirment 1: Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix, except in tester strain WP2uvrA in the presence of S9-mix
Mutation Expeirment 2: Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the background lawn and/or the presence of microcolonies, was observed in all tester strains, except in tester strain WP2uvrA
Mutation Expeirment 3: Cytotoxicity, as evidenced by a reduction of the background lawn and/or the presence of microcolonies, was observed in both the absence and presence of S9-mix at concentrations of 2500 μg/plate and upwards
Mutation Expeirment 4: Cytotoxicity, as evidenced by a reduction of the background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix.
Mutation Expeirment 5: Cytotoxicity, as evidenced by a decrease in the number of revertants, a reduction of the background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix.
Mutation Expeirment 6: The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

Additional information on mutation frequencies

Mutation experiment 1, 2 and 3 (DMSO):

- In the absence of S9-mix, the test item induced increases in tester strain TA98. Although the increases were above the historical control data range, the increases were not three-fold and therefore the increases are considered equivocal. In the presence of 10% (v/v) S9-mix, the test item induced increases in tester strain TA98. The increases observed were up to 3.1-fold compared to the concurrent vehicle control. However, the increases were within the historical control data range and therefore the increases are considered equivocal.

- All other bacterial strains showed negative responses over the entire dose range, i.e. no biologically relevant and/or significant dose-related increase (three-fold for TA1535 and TA1537 or two-fold for TA100 and WP2uvrA) in the number of revertants in any of the experiments.

- Since the test item is not stable in DMSO the first, second and third mutation experiment are considered invalid for the

evaluation of the mutagenic activity of the test item. Therefore, the experiments were repeated in the fourth, fifth and sixth experiment in the absence and presence of S9-mix in all five tester strains with anhydrous THF as solvent.

Mutation experiment 4, 5 and 6 (anhydrous THF):

- In the tester strains TA98 and TA1537, the test item induced up to 2.7- and 4.0-fold increases in the number of revertant colonies compared to the solvent control in the absence and presence of 10% (v/v) S9-mix, respectively. The increases observed were within the historical control data range and were only observed in one experiment. Therefore, these increases are not considered biologically relevant. In the other tester strains, no increase in the number of revertants was observed.

- The test item did not induce a biologically relevant and/or dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in the tester strain WP2uvrA both in the absence and presence of S9 metabolic activation in the fourth, fifth and sixth experiment.

Conclusions:
Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay when dissolved in a suitable vehicle (i.e. anhydrous THF).
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-08-29 to 2018-10-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Version / remarks:
July 29, 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: Mouse Lymphoma Assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: M16LB4523
- Expiration date of the lot/batch: 2018-11-17 (retest date)
- Purity test date: 2018-08-31

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: Not available
- Solubility and stability of the test substance in the solvent/vehicle: Not indicated
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not indicated

OTHER SPECIFICS: A correction factor of 1.00 for the purity/composition of the test item was applied in this study.
Target gene:
TK locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: American Type Culture Collection, (ATCC, Manassas, USA) (2001).
- Suitability of cells: L5178Y/TK+/--3.7.2C mouse lymphoma cells, recommended test system in international guidelines (e.g. OECD).
- Cell cycle length, doubling time or proliferation index: not indicated
- Methods for maintenance in cell culture if applicable: Stock cultures of the cells were stored in the freezer (-150°C). Cell density was kept below 1 x 10^6 cells/ml.
- Normal (negative control) cell cycle time: not indicated

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
Basic medium: RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin.
Growth medium: Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (R10 medium).
Exposure medium:
For 3 hour exposure: basic medium supplemented with 5% (v/v) heat-inactivated horse serum (R5-medium).
For 24 hour exposure: basic medium supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium).
Selective medium: basic medium supplemented with 20% (v/v) heat-inactivated horse serum (R20-medium) and 5 μg/ml trifluorothymidine (TFT) (Sigma).
Non-selective medium: non-selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (R20-medium).
Environmental conditions: All incubations were carried out in a humid atmosphere (80 - 100%, actual range 72 – 98%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.4 – 37.5°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Any variation to these conditions were evaluated and maintained in the raw data.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: not indicated
- Periodically 'cleansed' against high spontaneous background: yes, prior to dose range finding and mutagenicity testing, the mouse lymphoma cells were grown for 1 day in R10-medium containing 10^-4 M hypoxanthine, 2 x 10^-7 M aminopterine and 1.6 x 10^-5 M thymidine (HAT-medium) to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on R10 medium containing hypoxanthine and thymidine only. After this period cells were returned to R10-medium for at least 1 day before starting the experiment.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (rat liver metabolic activation system induced by a combination of phenobarbital and ß-naphthoflavone)
Test concentrations with justification for top dose:
Based on this solubility test, THF was selected as vehicle and 500 μg/ml as the maximum final concentration in culture medium (= 100 mg/ml in vehicle) for the dose range finding test.
Dose range finding test (3h treatment): 0, 31, 63, 125, 250 and 500 μg/mL with and without S9-mix;

Both in the absence and presence of S9-mix, no cell survival was observed at any of the test item concentrations. The test item precipitated in the exposure medium at all concentrations tested.
Since all dose levels showed severe cytotoxicity, an additional dose range finding test was performed with a 3 hour treatment period. Further investigation showed that at a concentration of 16 µg/ml the test item precipitated in the exposure medium.

Second dose range finding test (3h treatment): 0, 0.5, 1, 2, 4, 8 and 16 with and without S9-mix;
Second dose range finding test (24h treatment): 0, 0.5, 1, 2, 4, 8 and 16 without S9-mix;

The highest concentrations tested in the first mutation experiment were determined by the solubility of the test item in the culture medium.
The highest concentrations tested in the second mutation experiment were determined by the cytoxicity of the test item.
Mutagenicity assay I (3h treatment) without S9-mix: 0, 0.13, 0.25, 0.5, 1, 2, 4, 8, and 16 μg/ml;
Mutagenicity assay I (3h treatment) with S9-mix: 0, 0.13, 0.25, 0.5, 1, 2, 4, 8, and 12 μg/ml;
Mutagenicity assay II (24h treatment) without S9-mix: 0, 0.13, 0.5, 1, 2, 4, 8, 12 and 16 μg/ml.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: THF
- Justification for choice of solvent/vehicle: Based on a solubility test, THF was selected as vehicle and 500 μg/ml as the maximum final concentration in culture medium (= 100 mg/ml in vehicle) for the dose range finding test.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9-mix; at 15 μg/ml (3h treatment); at 5 μg/ml (24h treatment)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9-mix; at 7.5 μg/ml
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 8 x 10^6 cells (10^6 cells/ml for 3 hour treatment) or 6 x 10^6 cells (1.25 x 10^5 cells/ml for 24 hour treatment)

DURATION
- Exposure duration: 3h or 24h
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11 or 12 days
- Fixation time (start of exposure up to fixation or harvest of cells): 13-15 days

SELECTION AGENT (mutation assays): selective medium (TFT-selection)

STAIN (for cytogenetic assays): After the inclubation, the plates for the TFT-selection were stained for 1.5 - 2 hours, by adding 0.5 mg/ml 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to each well.

NUMBER OF REPLICATIONS:
1 replicate per concentration, the solvent control was tested in duplicate

NUMBER OF CELLS EVALUATED:
Determination of cloning efficiency: One cell was added per well (2 x 96-well microtiter plates/concentration) in non-selective medium.
Determination of mutation frequency: 9.6 x 10^5 cells/concentration plated in 5 x 96-well microtiter plates/concentration, each well containing 2000 cells in selective medium (TFT-selection); 9.6 x 10^5 cells/concentration plated (10 x 96-well microtiter plates/concentration), each well containing 1000 cells in selective medium (TFT-selection) (positive controls)
DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (RSG) (see calculations in section "any other information on materials and methods incl. tables")
Rationale for test conditions:
The highest concentration tested should give a relative total growth (RTG) of approximately 10-20% or should show a slight to heavy test item precipitation at the end of the treatment period or should correspond to 2 mg/ml or 0.01 M (whichever is the lowest).
The highest concentrations tested gave a relative total growth (RTG) of approximately 10-20% and the RTG in the lowest doses was approximately the same as the RTG in the solvent control. Some intermediate doses were also tested. At the highest concentration tested in the absence of S9-mix, also test item precipitate was observed.
Evaluation criteria:
In addition to the criteria stated below, any increase of the mutation frequency should be evaluated for its biological relevance including comparison of the results with the historical control data range.
The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
A test item is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. A Cochran Armitage trend test (p , 0.05) is performed to test whether there is a significant trend in the induction. Any observed increase should be biologically relevant and will be compared with the historical control data range.
A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test item is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
Statistics:
No statistical analysis
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: The pH and the osmolality of the culture medium containing the highest, non precipitating concentration were not determined.
- Water solubility: no data
- Precipitation: Mutagenicity assays: test item precipitation was observed at 16 μg/ml in the mutation experiments

RANGE-FINDING/SCREENING STUDIES:
First dose range finding experiment:
L5178Y mouse lymphoma cells were treated with a test item concentration range of 31 to 500 μg/ml in the absence and presence of S9-mix with a 3-hour treatment period.
Both in the absence and presence of S9-mix, no cell survival was observed at any of the test item concentrations. The test item precipitated in the exposure medium at all concentrations tested.
Since all dose levels showed severe cytotoxicity, an additional dose range finding test was performed with a 3 hour treatment period. Further investigation showed that at a concentration of 16 µg/ml the test item precipitated in the exposure medium.

Second dose range finding experiment:
L5178Y mouse lymphoma cells were treated with a test item concentration range of 0.5 to 16 μg/ml in the absence and presence of S9-mix with a 3-hour treatment period and in the absence of S9-mix with a 24-hour treatment period.
3h dose range finding experiment: The relative suspension growth was 3 and 11% at the test item concentration of 16 μg/ml compared to the relative suspension growth of the solvent control in the absence and presence of S9-mix, respectively. The test item precipitated in the exposure medium at 16 µg/ml.
24h dose range finding experiment: The relative suspension growth was 22% at the test item concentration of 16 μg/ml compared to the relative suspension growth of the solvent control. The test item precipitated in the exposure medium at 16 µg/ml.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.
- The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical solvent control database

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Mutation Experiment 1
In the absence of S9-mix, the dose level of 12 μg/ml was not used for mutation frequency measurement, since this dose level showed an inconsistent RSG compared to the concentrations of 8 and 16 μg/ml, i.e. no dose related toxicity was observed.
In the presence of S9-mix, the dose level of 16 μg/ml was not used for mutation frequency measurement, since this dose level was too toxic for further testing.
The dose levels selected to measure mutation frequencies at the TK-locus were:
Without S9-mix: 0.13, 0.25, 0.5, 1, 2, 4, 8 and 16 μg/ml exposure medium.
With S9-mix: 0.13, 0.25, 0.5, 1, 2, 4, 8 and 12 μg/ml exposure medium.
In the absence of S9-mix, the relative total growth of the highest test item concentration was 10% compared to the total growth of the concurrent solvent control.
In the presence of S9-mix, the relative total growth of the highest test item concentration was 14% compared to the total growth of the concurrent solvent controls.
Mutation Experiment 2
The dose levels of 0.13 to 1 μg/ml showed no cytotoxicity. Therefore, the dose level of 0.25 µg/ml was not regarded relevant for mutation frequency measurement.
The dose levels selected to measure mutation frequencies at the TK-locus were: 0.13, 0.5, 1, 2, 4, 8, 12 and 16 µg/ml exposure medium.
The relative total growth of the highest test item was 19% compared to the total growth of the concurrent solvent controls

OTHER:
Suspension growth: The suspension growth (SG) over the two-day expression period for cultures treated with THF was between 16 and 15 (3 hour treatment) and 77 and 94 (24 hour treatment)

List of deviations

1.  In the first mutation experiment in the absence of S9-mix, one solvent control was not used for the evaluation of the mutagenicity of the test item.

     Evaluation: The absolute cloning efficiency of one the solvent controls (CEday2)(135%) was above the acceptability criteria of the study (120%).In the revised OECD guideline 490, duplicate solvent control groups are no longer requested.Furthermore, the mutation frequency of the other solvent control was within the acceptability criteria and no increase in the mutation frequency above the GEF was observed in the treatment groups compared to the solvent control. Therefore, the determination of the mutagenicity of the test item with only one solvent control had no effect on the results of the study.

2.  The plates for the TFT-selection were stained with MTT for 1 hour and 40 minutes and 1 hour and 30 minutes in the first and second experiment, respectively.

     Evaluation: According to SOP GEN-H-508 (mutation test with mouse lymphoma L5178Y cells in micro wells) the plates should be stained for1.5 – 2 hours . Since the staining with MTT is only performed to enhance the contrast for colony counting, and the solvent and positive controls showed acceptable responses, this deviation has no effect on the study results.

 

The study integrity was not adversely affected by the deviations.

Conclusions:
In conclusion, the test item is mutagenic in the mouse lymphoma L5178Y test system in the presence of S9-mix at toxic dose levels. The test item is not mutagenic in the mouse lymphoma L5178Y test system in the absence of S9-mix.
Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-09-22 to 2018-06-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EU Method B.49 (In Vitro Mammalian Cell Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: M16LB4523
- Expiration date of the lot/batch: 2018-06-23 (retest date)
- Purity test date: 2018-04-17 (release date)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: No specific handling conditions required
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not indicated

OTHER SPECIFICS: correction factor of 1 for the purity/composition of the test item was applied
Target gene:
not applicable
Species / strain / cell type:
lymphocytes: cultured peripheral human lymphocytes
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: healthy adult, non-smoking volunteers (aged 18-35)
- Suitability of cells: stimulated human lymphocytes were used because they are sensitive indicators of clastogenic and aneugenic activity of a broad range of chemicals
- Sex, age and number of blood donors if applicable: ages 27, 32, 24, 30, 26 and 30
- Whether whole blood or separated lymphocytes were used if applicable: Blood samples were collected by venipuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started. Whole blood was used.
- Methods for maintenance in cell culture if applicable:
Lymphocyte cultures: Whole blood (0.4 ml) treated with heparin was added to 5 ml or 4.8 ml culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 ml (9 mg/ml) phytohaemagglutinin was added.
- Modal number of chromosomes: 46
- Normal (negative control) cell cycle time: AGT: 15.2h (age 27; dose-range finding study); 14.6h (age 32; first cytogenetic assay); 13.0h (age 24; cytogenetic assay 1A); 14.8h (age 30; cytogenetic assay 1B); 14.2h (age 26; second cytogenetic assay); 14.0h (age 30; cytogenetic assay 2A)

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
Culture medium: Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-in activated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 μg/mL respectively) and 30 U/mL heparin.
All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 40 - 89%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 34.7 - 37.1°C).
- Properly maintained: not applicable
- Periodically checked for Mycoplasma contamination: not applicable
- Periodically checked for karyotype stability: not applicable
- Periodically 'cleansed' against high spontaneous background: not applicable
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
Cytochalasin B
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-naphthoflavone induced rat liver microsomal enzymes (S9 homogenate)
Test concentrations with justification for top dose:
The highest tested concentration was determined by the precipitation of the test item observed in the culture medium.

Dose-range finding test: 15.6, 31.3, 62.5, 125, 250 and 500 μg/mL with S9-mix (3h treatment) and without S9-mix (3h and 24h treatment).

Based on the results of the dose range finding test an appropriate range of dose levels was chosen for the cytogenetic assays considering the highest dose level showed a cytotoxicity of 55 ± 5%  whereas the cytotoxicity of the lowest dose level was approximately the same as the cytotoxicity of the solvent control .

First cytogenetic assay:
With and without S9-mix: 10, 50, 100, 125, 150, 200 and 250 μg/ml culture medium (3 hour exposure time, 27 hour harvest time) (repeatead)

Cytogenetic assay 1A:
With and without S9-mix: 10, 50, 100, 125, 150, 200 and 250 μg/ml culture medium (3 hour exposure time, 27 hour harvest time) (repeated)

Cytogenetic assay 1B:
With and without S9-mix: 10, 50, 65, 80, 95, 110, 125, 140 and 155 μg/ml culture medium (3 hour exposure time, 27 hour harvest time)

Second cytogenetic assay
10, 30, 40, 50, 60 and 70 μg test item/ml culture medium (repeated)

Cytogenetic assay 2A:
10, 25, 50, 75, 100, 125 and 150 μg test item/ml culture medium.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: anhydrous THF (to which molecular sieve (0.4 nm) beads were added to absorb water before use)
- Justification for choice of solvent/vehicle:The test item was observed to be insoluble at 100 mg/ml in culture medium and ethanol. In DMSO, the test item was formed a homogenous suspension at 100 mg/ml. The test item precipitated in culture medium at the concentration of 50 mg/ml (= final concentration of 500 μg/ml) and above. Due to instability of the test item in DMSO, THF was selected as solvent in this study. An additional solubility determination was performed and showed the test item formed a clear yellow solution in THF at the concentration of 400 mg/ml (= final concentration of 2000 μg/ml in culture medium). The test item precipitated in culture medium at the concentration of 25 mg/ml (= final concentration of 250 μg/ml) and above. Based on these solubility findings, THF was selected as vehicle and 500 μg/ml was selected as maximum concentration for the dose range finding test.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
anhydrous THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9-mix; at 0.25 and 0.38 µg/mL (3h treatment); at 0.15 and 0.23 µg/mL (24h treatment)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
anhydrous THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Colchicine
Remarks:
without S9-mix; at 0.1 µg/mL (3h treatment); at 0.05 µg/mL (24h treatment)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
anhydrous THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9-mix; at 15 and 17.5 µg/mL (3h treatment)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
First cytogenetic assay
Lymphocytes were cultured for 46 ± 2 hours and thereafter exposed in duplicate to selected doses of the test item for 3 hours in the absence and presence of S9-mix. After 3 hours exposure, the cells were separated from the exposure medium by centrifugation. The supernatant was removed and the cells were rinsed once with 5 ml HBSS. After a second centrifugation step, HBSS was removed and cells were re-suspended in 5 ml culture medium with cytochalasin B (5 μg/ml) and incubated for another 24 hours.

Second cytogenetic assay
Lymphocytes were cultured for 46 ± 2 hours and thereafter exposed in duplicate to selected doses of the test item with cytochalasin B (5 μg/ml) for 24 hours in the absence of S9-mix. Appropriate vehicle and positive controls were included in the second cytogenetic assay.

DURATION
- Exposure duration: 3h (dose range finding test; first cytogenetic experiment) or 24h (dose range finding test; second cytogenetic experiment)
- Expression time (cells in growth medium): 24h (dose ranging test; first cytogenetic experiment) or no expression time (dose range finding test; second cytogenetic experiment)
- Fixation time (start of exposure up to fixation or harvest of cells): 27h (dose range finding test; first cytogenetic experiment) or 24h (dose range finding test; second cytogenetic experiment)

SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin B

STAIN (for cytogenetic assays): 5% (v/v) Giemsa

NUMBER OF REPLICATIONS: Duplicate

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
To harvest the cells, cell cultures were centrifuged and the supernatant was removed. Cells in the remaining cell pellet were resuspended in 1% Pluronic F68. After centrifugation, the cells in the remaining pellet were swollen by hypotonic 0.56% (w/v) potassium chloride solution. Immediately thereafter, ethanol/ acetic acid fixative (3:1 v/v) was added. Cells were collected by centrifugation and cells in the pellet were fixated carefully with 3 changes of ethanol / acetic acid fixative (3:1 v/v).

Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol / ether and cleaned with a tissue. The slides were marked with the Test Facility Study number and group number. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 - 30 min with 5% (v/v) Giemsa solution in Sörensen buffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded in a 1:10 mixture of xylene / pertex and mounted with a coverslip in an automated cover slipper.

NUMBER OF CELLS EVALUATED:
Three analysable concentrations were scored for micronuclei. At least 1000 (with a maximum deviation of 5%) binucleated cells per culture were examined by light microscopy for micronuclei. In addition, at least 1000 (with a maximum deviation of 5%) mononucleated cells per culture were scored for micronuclei separately. The number of micronuclei per cell was not recorded.
Since the lowest concentration of MMC-C and CP resulted in a positive response the highest concentration was not examined for the presence of micronuclei. Due to cytotoxicity the number of examined bi- or mononucleated cells in the positive control groups might be <1000. However, when an expected statistical significant increase is observed, this has no effect on the study integrity.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
The following criteria for scoring of binucleated cells were used:
- Main nuclei that were separate and of approximately equal size.
- Main nuclei that touch and even overlap as long as nuclear boundaries are able to be distinguished.
- Main nuclei that were linked by nucleoplasmic bridges.
The following cells were not scored:
- Trinucleated, quadranucleated, or multinucleated cells.
- Cells where main nuclei were undergoing apoptosis (because micronuclei may be gone already or may be caused by apoptotic process).
The following criteria for scoring micronuclei were adapted from Fenech, 1996:
- The diameter of micronuclei should be less than one-third of the main nucleus.
- Micronuclei should be separate from or marginally overlap with the main nucleus as long as there is clear identification of the nuclear boundary.
- Micronuclei should have similar staining as the main nucleus.

DETERMINATION OF CYTOTOXICITY
- Method: The cytostasis / cytotoxicity was determined by calculating the Cytokinesis-Block Proliferation Index (CBPI).
- Any supplementary information relevant to cytotoxicity: A minimum of 500 cells per culture was counted, scoring cells with one, two or more nuclei (multinucleated cells).
Rationale for test conditions:
The highest concentration examined for micronuclei should be cultures that produce 55 ± 5% of cytotoxicity. The lowest concentration should have little or no cytotoxicity (approximately the same as vehicle control). Also cultures treated with an intermediate concentration should be examined. For poorly soluble test items, the highest concentration examined should be the lowest insoluble concentration determined at the end of treatment irrespective of toxicity. The extent of precipitation may not interfere with the scoring of micronuclei. If no precipitate or limiting toxicity is observed, the highest concentration examined should correspond to 2 mg/ml or 0.01 M, whichever is the lowest.
Evaluation criteria:
A test item is considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if all of the following criteria are met:
a) At least one of the test concentrations exhibits a statistically significant (Fisher exact test, one-sided, p < 0.05) increase compared with the concurrent vehicle control.
b) The increase is dose-related in at least one experimental condition when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical vehicle control data range.

A test item is considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
a) None of the test concentrations exhibits a statistically significant (Fisher exact test, one-sided, p < 0.05) increase compared with the concurrent vehicle control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are inside the 95% control limits of the historical vehicle control data range.
Statistics:
Fisher exact test was used to identify results significantly different from control group.
Species / strain:
lymphocytes: cultured peripheral human lymphocytes
Remarks:
first and second cytogenetic experiments primary assays
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see additional information on results
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Water solubility: No data
- Precipitation :
Cytogenetic Assay 1B: The test item precipitated at the concentration of 155 μg/ml.
Cytogenetic Assay 2A The test item precipitated in the culture medium at the concentration of 150 μg/ml.

RANGE-FINDING/SCREENING STUDIES:
In order to select the appropriate dose levels for the in vitro micronucleus test cytotoxicity data was obtained in a dose range finding test: blood cultures were treated with 15.6, 31.3, 62.5, 125, 250 and 500 μg test item/ml culture medium and exposed for 3 and 24 hours in the absence of S9-mix and for 3 hours in the presence of S9-mix.
Dose-related cytotoxicity was observed after 3 hours of exposure at 125 μg/ml culture medium and above in the absence and presence of S9-mix, respectively and after 24 hours of exposure at 62.5 μg/ml culture medium and above. The test item precipitated in the culture medium at the concentrations of 250 μg/ml and above.

NUMBER OF CELLS WITH MICRONUCLEI
The test item did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two experiments.

HISTORICAL CONTROL DATA
- Positive historical control data: The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. Furthermore, colchicine also showed a statistically significant increase in the number of binucleated cells with micronuclei in the second cytogenetic assay. In addition, the number of mono- and binucleated cells with micronuclei found in the positive control cultures as within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.
- The number of mono- and binucleated cells with micronuclei found in the solvent control was within the 95% control limits of the distribution of the historical vehicle control database.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: CPBI index (cytokinesis-block proliferation index);

In cytogenetic assay 1B, the test item was tested up to 155 μg/ml for a 3 hour exposure period with a 27 hour harvest time in the absence and presence of S9-mix. Appropriate toxicity was reached at 140 μg/ml culture medium in the absence of S9-mix (cytostasis/cytotoxicity of 52% compared to concurrent vehicle control) and in the presence of S9-mix (cytostasis/cytotoxicity of 54% compared to concurrent vehicle control).
Therefore the following dose levels were selected for scoring of micronuclei:
Without S9-mix: 10, 125 and 140 μg/ml culture medium (3 hour exposure time, 27 hour harvest time)
With S9-mix : 10, 110 and 140 μg/ml culture medium (3 hour exposure time, 27 hour harvest time)

In cytogenetic assay 2A, the test item was tested up to 150 μg/ml for a 24 hour exposure period with a 24 hour harvest time in the absence of S9-mix. Appropriate toxicity was reached at 75 μg/ml culture medium (cytostasis/cytotoxicity of 49% compared to concurrent vehicle control).
Therefore the following dose levels were selected for the scoring of micronuclei: 10, 50 and 75 μg test item/ml culture medium.
Conclusions:
Based on the results of this study, it is concluded that the test item is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described in this report.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Bacterial reverse mutation assay

A key study (K1, Gijsbrechts, J, 2018) was performed according to OECD Guideline 471 (Bacterial Reverse Mutation Assay).Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 were treated with solutions of the test item using the Ames plate incorporation method at 9 dose levels, in triplicate, both with and without the addition of liver S9 in standard co-factors.

In this study, acceptable responses were obtained for the vehicle and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Since the test item is not stable in dimethyl sulfoxide, the first, second and third mutation experiment are considered invalid for the evaluation of the mutagenic activity of the test item. Therefore, the experiments were repeated in the fourth, fifth and sixth experiment in the absence and presence of S9-mix in all five tester strains with anhydrous tetra hydrofuran as solvent.

The test item did not induce a biologically relevant and/or dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in the tester strain WP2uvrA both in the absence and presence of S9 metabolic activation in the fourth, fifth and sixth experiment.

In vitro micronucleus assay

In an in vitro micronucleus assay in cultured peripheral human lymphocytes (Eurlings, 2018), performed according to OECD Guideline 487 (In Vitro Mammalian Cell Micronucleus Test), the test substance was assessed for its potential to induce micronuclei formation in cultured peripheral human lymphocytes in the presence of S9-mix rat liver metabolic activation system induced by a combination of phenobarbital and ß-naphthoflavone) with a 3 hour exposure period and in the absence of S9-mix with a 3 and 24 hour exposure period. The test item was dissolved in anhydrous tetrathydrofuran (THF).

In the first cytogenetic assay, the test item was tested up to 140 μg/ml for a 3-hour exposure period with a 27 hour harvest time in the absence and presence of S9-mix. Appropriate toxicity (reduction in the cytokinesis-block proliferation index to 45 ± 5%) was reached at this dose level.

In the second cytogenetic assay, the test item was tested up to 75 μg/ml for a 24 hour exposure period with a 24 hour harvest time in the absence of S9-mix. Appropriate toxicity (reduction in the cytokinesis-block proliferation index to 45 ± 5%) was reached at this dose level.

The number of mono- and binucleated cells with micronuclei found in the solvent control cultures was within the 95% control limits of the distribution of the historical vehicle control database. The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated and binucleated cells with micronuclei. In addition, the number of mono- and binucleated cells with micronuclei found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

The test item did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two experiments.

It is concluded that this test is valid and that the test item is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described in this report.

 

In vitro gene mutation study in mammalian cells:

Gijsbrechts (2018) investigated the mutagenic activity of the test item in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells.

The test item, dissolved in anhydrous tetrahydrofuran (THF), was assessed for its potential to induce forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells. The test was performed in the presence of S9-mix with a 3-hour treatment period and in the absence of S9-mix with a 3 and 24-hour treatment period.

In the first mutation experiment, the test item was tested up to concentrations of 16 and 12 µg/ml in the absence and presence of S9-mix, respectively. The treatment period was 3 hours. The Relative Total Growth (RTG) at the highest dose level was 10% and 14% in the absence and presence of S9-mix, respectively. The test item precipitated in the culture medium at the concentration of 16 µg/ml.

In the second mutation experiment, the test item was tested up to concentrations of 16 µg/ml in the absence of S9-mix. The treatment period was 24 hours. The Relative Total Growth (RTG) was 19% at the concentration of 16 µg/ml. The test item precipitated in the culture medium at the concentration of 16 µg/ml.

The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical solvent control database.

Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

In the absence of S9-mix, the test item did not induce a significant increase in the mutation frequency after a 3 hour treatment period. This result was confirmed in an independent experiment with a treatment duration of 24 hours.

In thepresence of S9-mix, the test item induced an up to 4.0-fold increase in the mutation frequency. The increase was above the 95% control limits of the distribution of the historical negative control database and also above the Global Evaluation Factor

(GEF) + MF(controls) (191 per 10^6survivors) at the dose level of 12 μg/ml. No increase above the GEF was observed at the lower cytotoxic dose level of 8 µg/ml (RTG of 48%).

In addition, a statistically significant dose related trend (p<0.001) was observed in the presence of S9-mix.

According to the OECD 490 guideline care should be taken when interpreting positive results only found between 20 and 10% RTG.

Although the increase in the mutation frequency at the TK locus in the presence of
S9-mix was only observed at a toxic dose level (RTG 14%), the mutation frequency at this concentration was above the GEF and a significant dose-related trend was observed in the induction (p<0.001). Therefore, the result is considered to be biological relevant and the test item mutagenic at toxic dose levels

Justification for classification or non-classification

Positive results were observed in the in vitro mouse lymphoma assay, and negative results were observed in the in vitro bacterial reverse mutation assay and the in vitro mammalian cell micronucleus test.

In order to implement appropriate risk management measures for substances potentially meeting the criteria for the highest risk categories 1A, 1B or 2 for genotoxicity, the test item is classified according to a worst case scenario without doing any further in vivo testing.

Based on the available data, the criteria laid down in the CLP Regulation and the precautionary principle, the test item 4 -nitrobenzene-1 -sulfonyl chloride should be classified as Repr 2 - H360: May damage fertility or the unborn child and

Muta 2 - H341 Suspected of causing genetic defects.