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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

OECD 471, analogue substance, 2016, positive

modified OECD 471, analogue substance, 2020, positive

OECD 487. in vitro micronucleo, 2016, negative

OECD 476, in vitro HPRT, 2016, negative

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
other: experimental data on similar substance
Adequacy of study:
key study
Study period:
2015-11-30 to 2016-05-04
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
28 Jul 2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
30 May 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
Aug 1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: in vitro gene mutation test in mammalian cells
Target gene:
HPRT locus
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CELL LINE AND STORAGE
The CHO (Chinese hamster ovary) cell line is a permanent cell line derived from the Chinese hamster and has a
- high proliferation rate (doubling time of about 12 - 16 hours)
- high plating efficiency (about 90 %)
- karyotype with a modal number of 20 chromosomes.
Stocks of the CHO cell line (1-mL portions) are maintained at -196 °C in liquid nitrogen using 7 % (v/v) DMSO in culture medium as a cryoprotectant. Each batch used for mutagenicity testing was checked for mycoplasma contamination.

CULTURE MEDIA
- All media were supplemented with 1 % (v/v) penicillin/streptomycin (stock solution: 10000 IU / 10000 μg/mL) and 1 % (v/v) amphotericine B (stock solution: 250 μg/mL)
- Culture medium for the 1st Experiment: Ham's F12 medium containing stable glutamine and hypoxanthine (Biochrom; Cat. No. FG 0815) supplemented with 10 % (v/v) fetal calf serum (FCS).
- Culture medium for the 2nd Experiment: Ham's F12 medium containing stable glutamine and hypoxanthine (PAN Biotech; Cat. No.P04-15500) supplemented with 10 % (v/v) fetal calf serum (FCS).
- Treatment medium (without S9 mix): Ham's F12 medium containing stable glutamine and hypoxanthine supplemented with 10 % (v/v) FCS.
- Treatment medium (with S9 mix): Ham's F12 medium containing stable glutamine and hypoxanthine.
- Pretreatment medium ("HAT" medium): Ham's F12 medium supplemented with hypoxanthine (13.6 x 1E-3 mg/mL), aminopterin (0.18 x 1E-3 mg/mL), thymidine (3.88 x 1E-3 mg/mL) and 10% (v/v) FCS
- Selection medium ("TG" medium): Ham's F12 medium containing stable glutamine and hypoxanthine supplemented with 6-thioguanine (10 μg/mL) and 10 % (v/v) fetal calf serum (FCS)

CELL CULTURE
For cell cultivation, deep-frozen cell suspensions were thawed at 37 °C in a water bath, and volumes of 0.5 mL were transferred into 25 cm² plastic flasks containing about 5 mL Ham's F12 medium including 10 % (v/v) FCS. Cells were grown with 5 % (v/v) CO2 at 37 °C and ≥ 9 0 % relative humidity up to approximate confluence and subcultured twice weekly (routine passage in 75 cm² plastic flasks).

ROUTINE PASSAGE (preparation of a single cell suspension in 75 cm² flask)
- Cell medium was removed and cells were washed with 5 mL PBS or HBSS (both Ca-Mg-free).
- Cells were trypsinized with 2 mL HBSS (Hanks balanced salt solution; Ca-Mg-free) and 2 mL trypsin (0.25 % [w/v]) to remove the cells from the bottom of the plastic flasks.
- This reaction was stopped by adding 6 mL culture medium incl. 10 % (v/v) FCS.
- Cells were pipetted up and down to separate them and to prepare a homogeneous single cell suspension.
- Cells were counted in a counting chamber or using a cell counter.
- Cell suspensions were diluted with complete culture medium to the desired cell count.
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction from phenobarbital (i.p.) and β-naphthoflavone (oral) induced Wistar rat livers
Test concentrations with justification for top dose:
PRETEST:
- Justification: Following the requirements of the current international guidelines and the ICPEMC Task Group (5) a test substance should be tested up to a maximum concentration of 2 mg/mL, 2 μL/mL or 10 mM, whichever is the lowest. In case of toxicity, the top dose should result in approximately 10 - 20 % relative survival (relative cloning efficiency), but not less than 10%. For relatively insoluble test substances at least one concentration should be scored showing no precipitation in culture medium at the end of the exposure period. In the pretest for toxicity based on the purity of the test substance 6500.0 μg/mL was used as top concentration.
- concentrations with and without S9 mix [4 hour exposure]: 25.4, 50.8, 101.6, 203.1, 406.3, 812.5, 1625.0, 3250.0, 6500.0 µg/mL

MAIN TESTS
- justification: Doses were based on the data and the observations from the pretest and current guidelines were taking into account.
- concentrations Experiment 1, with and without S9 mix [4 hour exposure]: 2.5, 5.0, 10.0, 20.0, 40.0, 80.0, 160.0 µg/mL
- concentrations Experiment 2, with and without S9 mix [4 hour exposure]: 6.3, 12.5, 25.0, 50.0, 100.0, 160.0 µg/mL
Vehicle / solvent:
PRETEST
- Vehicle: culture medium
- Justification for choice of vehicle: it was the only feasible vehicle to reach a maximum concentration of 6500 μg/mL (homogeneous suspension) due to physico-chemical properties.

MAIN TEST
- Vehicle: DMSO
- Justification for choice of vehicle: in the pretest, precipitates were found in all exposed test groups from the lowest applied concentration of 25.4 μg/mL onward. DMSO was chosen based on an additional solubility test with a lower top concentration.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Test substance preparation: The substance was dissolved in DMSO. The test substance was weighed and topped up with the chosen vehicle to achieve the required concentration of the stock solution. To achieve a solution of the test substance in the vehicle, the test substance preparation was shaken thoroughly. The further concentrations were diluted according to the planned doses. All test substance solutions were prepared immediately before administration.
- Preparation of test cultures: Cell stocks (1.0-mL portions) stored in liquid nitrogen were thawed at 37 °C in a water bath. 0.5 mL of stock cultures were pipetted into 25 cm² plastic flasks containing 5 mL Ham's F12 medium (incl. 10 % [v/v] FCS). After 24 hours, the medium was replaced to remove any dead cells. At least 2 passages were performed before cells were taken for the experiment. A further passage was also necessary in order to prepare test cultures.
- Pretreatment of cells with "HAT" medium: During the week prior to treatment, any spontaneous HPRT-deficient mutants were eliminated by pretreatment with "HAT" medium. 3 – 5E+5 cells were seeded per flask (75 cm²) and incubated with "HAT" medium for 3 - 4 days. A subsequent passage in Ham's F12 medium incl. 10 % (v/v) FCS was incubated for a further 3 - 4 days.
- Attachment period: For each test group, about 20E+6 logarithmically growing cells per flask (300 cm²) were seeded into about 40 mL Ham's F12 medium supplemented with 10 % (v/v) FCS and incubated for about 20 - 24 hours.
- Exposure period: After the attachment period, the medium was removed from the flasks and the treatment medium was added. The cultures were incubated for the respective exposure period at 37 °C, 5 % (v/v) CO2 and ≥ 90 % relative humidity.
- Expression period: The exposure period was completed by rinsing several times with HBSS. This was directly followed by the 1st passage in which 2E+6 cells were seeded in 20 mL medium (in 175 cm² flasks). The flasks were left to stand in the incubator for about 3 days at 37 °C, relative humidity of ≥ 90 % and 5 % (v/v) CO2 atmosphere. After about 3 days, the cells were passaged a 2nd time in 175 cm² flasks with 2E+6 cells. After the expression period the cells were transferred into selection medium (3rd passage).
- Selection period: For selection of the mutants, two 175 cm² flasks with 2E+6 cells each from every treatment group, if possible, were seeded in 20 mL selection medium ("TG" medium) at the end of the expression period. The flasks were returned to the incubator. At the end of the selection period, the medium was removed and the remaining colonies were fixed with methanol, stained with Giemsa and counted.

DURATION
- Preincubation period: 20 - 24 hours after seeding
- Exposure duration: 4 hours
- Expression time: 7 – 9 days
- Selection time: 6 – 7 days
- Fixation time: from day 16

SELECTION AGENT: TG medium

STAIN: Giemsa

NUMBER OF REPLICATIONS: 2

CYTOTOXICITY DETERMINATION
- Cloning efficiency (CE) (pre-experiment): The determination of the cloning efficiency in the pre-experiment was similar to that described for the determination of the cloning efficiency 1 (CE1) in the main experiments, excepting that 1E+6 cells were seeded in 25 cm² flasks coated with 5 mL Ham´s F12 medium incl. 10 % (v/v) FCS. After test substance incubation, 200 cells were transferred into new Ham´s F12 medium incl. 10 % (v/v) FCS. Due to technical reasons in the pretest no cytotoxicity data were obtained.
- Cloning efficiency 1 (CE1; survival): For the determination of the influence of the test substance after the exposure period, about 200 cells per concentration were reserved from the treated cells and were seeded in 25 cm² flasks and coated with 5 mL Ham's F12 medium incl. 10% (v/v) FCS in parallel to the 1st passage directly after test substance incubation.
- Cloning efficiency 2 (CE2; viability): For the determination of the mutation rate after the expression period, two aliquots of about 200 cells each were reserved from the transfer into selection medium (after 7 – 9 days) and seeded in two flasks (25 cm2) containing 5 mL Ham's F12 medium incl. 10% (v/v) FCS. In all cases, after seeding the flasks were incubated for 5 - 8 days to form colonies. These colonies were fixed, stained and counted. The absolute and relative cloning efficiencies (%) were calculated for each test group

CHECK OR DETERMINATION OF FURTHER PARAMETERS
- pH: the pH was measured at least for the top concentrations and for the vehicle controls with and without S9 mix.
- Osmolality: Osmolality was measured in at least the top concentrations and the vehicle controls with and without S9 mix.
- Solubility: Test substance precipitation was assessed immediately after dosing the test cultures and at the end of treatment.
- Cell morphology: The test cultures of all test groups were examined microscopically for cell morphology and cellular attachment at the end of the exposure period, which is a further indication for cytotoxicity.
Evaluation criteria:
ACCEPTANCE CRITERIA
The HPRT assay is considered valid if the following criteria are met:
- The absolute cloning efficiencies of the negative/vehicle controls should not be less than 50 % (with and without S9 mix).
- The background mutant frequency in the negative/vehicle controls should be within our historical negative control data range (95 % control limit). Weak outliers can be judged acceptable if there is no evidence that the test system is not “under control”.
- The positive controls both with and without S9 mix should induce a distinct, statistically significant increase in mutant frequencies in the expected range.

ASSESSMENT CRITERIA
A test substance is considered to be clearly positive if all following criteria are met:
- A statistically significant increase in mutant frequencies is obtained.
- A dose-related increase in mutant frequencies is observed.
- The corrected mutation frequencies (MFcorr.) exceeds both the concurrent negative/vehicle control value and the range of the laboratory’s historical negative control data (95 % control limit).
Isolated increases of mutant frequencies above our historical negative control range or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity. A test substance is considered to be clearly negative if the following criteria are met:
- Neither a statistically significant nor dose-related increase in the corrected mutation frequencies is observed under any experimental condition.
- The corrected mutation frequencies in all treated test groups is close to the concurrent vehicle control value and within the range of the laboratory’s historical negative control data (95% control limit).
Statistics:
- An appropriate statistical trend test (MS EXCEL function RGP) was performed to assess a possible dose-related increase of mutant frequencies. The used model is one of the proposed models of the International Workshop on Genotoxicity Test procedures Workgroup Report. The dependent variable was the corrected mutant frequency and the independent variable was the concentration. The trend was judged as statistically significant whenever the one-sided p-value (probability value) was below 0.05 and the slope was greater than 0.
- In addition, a pair-wise comparison of each test group with the vehicle control group was carried out using one-sided Fisher's exact test with Bonferroni-Holm correction. The calculation was performed using R.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
Osmolality and pH values were not influenced by test substance treatment.

RANGE-FINDING/SCREENING STUDIES:
In the pretest the pH value was not influenced by the addition of the test substance preparation to the culture medium at the concentrations measured. In addition, a homogeneous suspension of the test substance in the vehicle HAM´s F12 was obtained in the stock preparation (Test group: 6500 μg/mL) as well as in all applied concentrations down to 25.4 μg/mL. Thus, no soluble test substance concentration was obtained. Due to techniqual reasons, only the results of the cell counting during the 1st passage after test substance incubation could be used for dose selection.

CELL MORPHOLOGY
Only in the 1st Experiment in the absence of S9 mix, after 4 hours treatment in the morphology and attachment of the cells was adversely influenced (grade > 2) in the highest applied concentration (160.0 μg/mL).

MUTANT FREQUENCY
In this study, no relevant increase in the number of mutant colonies was observed with or without S9 mix. In both experiments after 4 hours treatment with the test substance the values for the corrected mutation frequencies (MFcorr.: 0.00 – 9.79 per E+6 cells) were close to the respective vehicle control values (MFcorr.: 1.44 – 8.29 per E+6 cells) and close to or within the range of the 95% control limit of our historical negative control data (MFcorr.: 0.00 – 6.84 per E+6 cells). However, in the 1st Experiment in the presence of S9 mix the values for the corrected mutation frequencies in test group 40.0 μg/mL (MFcorr.: 8.89 per E+6 cells) and 160 μg/mL (MFcorr.: 9.79 per E+6 cells) were slightly above the range of the 95% control limit and slightly above the concurrent vehicle control (MFcorr.: 8.29 per E+6 cells). Nevertheless, the values were neither statistically significant nor dose-related increased. In addition, the results obtained in the 1st Experiment in the presence of S9 mix could not be confirmed in the 2nd Experiment.
In all experiments, no statistically significant dose-related increase in the mutant frequency was found in cells after 4 hours of treatment either in the absence or presence of S9 mix.
However, in the 1st Experiment in the absence of S9 mix the values for the corrected mutation frequencies in test group 40.0 μg/mL (MFcorr.: 6.27per E+6 cells) and 80 μg/mL (MFcorr.: 7.75 per E+6 cells) were statistically significant compared with the respective vehicle control (MFcorr.: 1.44 per E+6 cells). Nevertheless, the values obtained for the corrected mutation frequency of this experimental part were close to the range of the 95% control limit and well within our historical negative control data range (MFcorr.: 0.00 – 9.16 per E+6 cells). Therefore, this finding has to be regarded as biologically irrelevant.
The positive control substances EMS (without S9 mix; 400 μg/mL) and DMBA (with S9 mix; 1.25 μg/mL) induced a clear increase in mutation frequencies, as expected. The values of the corrected mutant frequencies (without S9 mix: MFcorr.: 42.47 – 264.00 per E+6 cells; with S9 mix: MFcorr.: 96.88 – 189.14 per E+6 cells) were within our historical positive control data range (without S9 mix: MFcorr.: 62.02 – 268.09 per E+6 cells; with S9 mix: MFcorr.: 41.99 – 736.50 per E+6 cells).

Cytotoxic effects, as indicated by clearly reduced cloning efficiencies of about or below 20 % of the respective negative control values were not observed in both experiments in the presence and absence of S9 mix, up to the highest applied concentrations.

Conclusions:
Negative with and without metabolic activation.
Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
other: experimental data on similar substance
Adequacy of study:
key study
Study period:
2015-11-17 to 2016-05-18
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
26 Sep 2014
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: In vitro Mammalian Cell Micronucleus Test, No B.49; No L 193
Version / remarks:
Commission Regulation (EC) No 640/2012 of 06 July 2012
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CELL LINE AND STORAGE
The V79 cell line is a permanent cell line derived from the Chinese hamster and has a
− high proliferation rate (doubling time of about 12 - 14 hours),
− high plating efficiency (≥ 90 %),
− stable karyotype (modal number of 22 chromosomes).
The V79 cell line has shown its suitability to detect aneugenic effects in the Micronucleus test in vitro either in the absence and presence of cytochalasin B.
Stocks of the V79 cell line (1-mL portions) were maintained at -196 °C in liquid nitrogen using 7 % (v/v) dimethyl sulfoxide (DMSO) in culture medium as a cryoprotectant. Each batch used for the cytogenetic experiments was checked for
− mycoplasma contamination,
− karyotype stability,
− plating efficiency (=colony forming ability) incl. vital staining.

CULTURE MEDIA
MEM (minimal essential medium with Earle's salts) containing a L-glutamine source supplemented with
− 10 % (v/v) fetal calf serum (FCS)
− 1 % (v/v) penicillin/streptomycin (10000 IU / 10000 μg/mL)
− 1 % (v/v) amphotericine B (250 μg/mL)

CELL CULTURE
Deep-frozen cell stocks were thawed at 37 °C in a water bath, and volumes of 0.5 mL were transferred into 25 cm² plastic flasks containing about 5 mL MEM supplemented with 10 % (v/v) FCS. Cells were grown with 5 % (v/v) CO2 at 37 °C and ≥ 90 % relative humidity and subcultured twice weekly. Cell monolayers were suspended in culture medium after detachment with 0.25 % (w/v) trypsin solution.

CELL CYCLE AND HARVEST TIME
The cell cycle of the untreated V79 cells lasts for about 12 - 14 hours under the selected culture conditions (last measurement based on the BrdU method of Speit et al.: 12 hours; May 2014). Thus, a harvest time of 24 hours is about 2 times the normal cell cycle length.
V79 cells are an asynchronous cell population, i.e. at the time of test substance treatment there are different cell stages (G1-, S-, G2-phase and mitosis). Since the effect on these cell stages may vary for different test substances, more than one harvest time after treatment may be appropriate.
Furthermore, substance-induced mitotic delay may considerably delay the first post-treatment mitosis. Therefore, delayed harvest times (e.g. 44 hours) and prolonged exposure periods (e.g. 24 hours treatment) may be required for the detection of several substances.
Cytokinesis block (if used):
actin polymerisation inhibitor cytochalasin B
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction from phenobarbital and β-naphthoflavone induced Wistar rat livers
Test concentrations with justification for top dose:
PRETEST
- Justification: Following the requirements of the current OECD Guideline 487 a test substance with defined composition should be tested up to a maximum concentration of 2 mg/mL, 2 μL/mL or 10 mM, whichever is the lowest. When the test substance is not of defined composition, e.g. substance of unknown or variable composition, complex reaction products or biological materials (socalled UVCBs), or environmental extracts, the top concentration should be higher to increase the concentration of each of the components (e.g. 5 mg/mL). In case of toxicity, the top concentration should produce 55 ± 5 % cytotoxicity: reduction of the proliferation index (CBPI) to 45 ± 5 % of the concurrent vehicle control. For relatively insoluble test substances only one concentration should be tested showing turbidity or precipitation in culture medium at the end of exposure period. In the pretest for toxicity based on the content of the Cr-complex of the test substance in the 2600 μg/mL total substance was used as top concentration.
- Concentrations, with and without S9 mix: 20.3, 40.6, 81.3, 162.5, 325.0, 650.0, 1300.0, 2600.0 μg/mL

MAIN TEST
- Justification: The concentrations tested in this study were selected in accordance with the requirements set forth in the test guidelines and based on the results of a preliminary range finding test (experimental conduct with records and documentation in general accordance with the GLP principles).
- Concentrations 1ST Experiment, with and without S9 mix, 24 hour preparation interval: 5.00, 10.00, 20.00, 40.00, 80.00, 160.00 μg/mL
- Concentrations 2ST Experiment, without S9 mix, 24 hour preparation interval: 10.00, 20.00, 40.00, 80.00, 160.00 μg/mL
- Concentrations 2ST Experiment, with and without S9 mix, 44 hour preparation interval: 10.00, 20.00, 40.00, 80.00, 160.00 μg/mL
Vehicle / solvent:
PRETEST
- Vehicle used: culture medium
- Justification for choice of vehicle: Culture medium was the only feasible vehicle to reach a maximum concentration of 2600 μg/mL (homogeneous suspension) due to physico-chemical properties.

MAIN TEST
- Vehicle used: DMSO
- Justification for choice of vehicle: In the pretest, precipitates were found in all exposed test groups from the lowest applied concentration of 20 μg/mL onward. Following an additional solubility test with a lower top concentration it was decided to use dimethyl sulfoxide (DMSO) as vehicle in the main experiments of this study.
Untreated negative controls:
yes
Remarks:
without vehicle and S9 mix
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
EXPERIMENTAL PROCEDURE
- Method of application: in medium
- Preparation of test cultures: The stocks of cells (1.0-mL portions) were thawed at 37 °C in a water bath. 0.5 mL were pipetted into 25 cm² cell culture flasks containing 5 mL MEM (incl. 10 % [v/v] FCS). The flasks were subsequently incubated at 37 °C, 5 % (v/v) CO2 and relative humidity of ≥ 90 % until they have reached confluency of at least 50 % (duration about 2 – 4 days). The medium was replaced after about 24 - 30 hours to remove any dead cells. Prior to the preparation of the final test cultures, the cells may run through max. 15 routine passages. After the "last" routine passage, there was another passage to prepare test cultures.
- Seeding of the cells: A single cell suspension with the required cell count (3 - 5 E+5 cells per culture, depending on the schedule) was prepared in MEM incl. 10 % (v/v) FCS. 5 mL cell suspension was transferred into 25 cm² cell culture flasks using a dispenser. Subsequently, the test cultures were incubated at 37 °C, 5 % (v/v) CO2 and ≥ 90 % relative humidity. The cultures were visually checked for attachment and viability before treatment of the test cultures.
- Definition of test cultures: A test group consists of two separately treated flasks (Culture A and B). Each, two slides were prepared and, thus, four slides were available for scoring of each test group, in general.
- Treatment of test cultures: After the attachment period, about 20 - 24 hours after seeding, the medium was removed from the flasks and the treatment medium was added. The cultures were incubated for the respective exposure period at 37 °C, 5 % (v/v) CO2 and ≥ 90 % relative humidity.
- At the end of the 4-hour exposure period, the medium was removed and the cultures were rinsed twice with 5 mL HBSS (Hanks Balanced Salt Solution). Subsequently, 5 mL MEM (incl. 10 % [v/v] FCS) supplemented with CytB (final concentration: 3 μg/mL; stock: 0.6 mg/mL in DMSO; AppliChem, Cat.No. A7657) was added and the cultures were incubated at 37 °C, 5 % (v/v) CO2 and ≥ 90 % relative humidity for the respective recovery time. In the case of 24-hour continuous exposure, CytB was added to the treatment medium at start of treatment, and cell preparation was started directly at the end of exposure. At 44 hours preparation interval in the presence of S9 mix CytB was added 24 hours before preparation of the cultures.

DURATION
Exposure-, Recovery- and Harvest time: see Table 1.

SPINDLE INHIBITOR
actin polymerisation inhibitor cytochalasin B (CytB)

STAIN
4’,6-diamidino-2-phenylindole dihydrochloride and propidium iodide

NUMBER OF REPLICATIONS
2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED
- Cell harvest and preparation of slides: The cells were prepared based on the method described by Fenech, 1993. Just before preparation the culture medium was completely removed. Single cell suspensions were prepared from each test group by enzymatic dissociation. Then, the cell number per flask of each cell suspension was determined using a cell counter (CASY®, Roche Applied Science, Mannheim, Germany). Subsequently, 5xE+4 cells per slide were centrifuged at 600 rpm for 7 minutes onto labelled slides using a Cytospin centrifuge (Cellspin I, Tharmac, Waldsolms, Germany). At least two slides per flask were prepared. In the case of strongly reduced cell numbers below 10x1E4 cells per flask no slides were prepared. After drying, the slides were fixed in 90 % (v/v) methanol for 10 minutes.
- Staining: Before scoring, the slides were stained with a mixture of 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI; stock: 5 mg/mL; Sigma-Aldrich, Cat.No. D9542) and propidium iodide (stock: 5 mg/mL; Sigma-Aldrich, Cat.No. P4170) in Fluoroshield™ (Sigma-Aldrich, Cat.No. F6182) at a concentration of 0.25 μg/mL each. By the use of the combination of both fluorescence dyes it can be differentiated between DNA (DAPI; excitation: 350 nm, emission: 460 nm) and cytoplasm (PI; excitation: 488 nm, emission: 590 nm).

NUMBER OF CELLS EVALUATED
At least 1000 binucleated cells per culture, in total at least 2000 binucleated cells per test group

CRITERIA FOR MICRONUCLEUS IDENTIFICATION
see 'Evaluation criteria'.

DETERMINATION OF CYTOTOXICITY
- Method: relative population doubling (RPD), Proliferation Index (CBPI)
- Any supplementary information relevant to cytotoxicity (RPD): The RPD takes into account either cytotoxicity or cell proliferation over the whole incubation period until slide preparation when determination of cell numbers occurs. However, in the main experiments supplementation of culture medium with CytB blocks cell division. Thus, under the experimental conditions described, RPD is an indication of cell viability mainly for the time period before addition of CytB.

OTHER EXAMINATIONS:
- Cell morphology: At the end of the treatment period, all test groups were examined microscopically with regard to cell morphology, which is a further indication for cytotoxicity.
- pH value: Changes in the pH were apparent by a color change of the indicator in the culture medium (phenol red: normal range: about pH 6.7 - 8.3). The pH was measured at least for the top concentration and for the vehicle control with and without S9 mix.
- Osmolality: Osmolality was measured at least for the top concentration and for the vehicle control with and without S9 mix.
- Solubility: Test substance precipitation was checked immediately after start of treatment of the test cultures (macroscopically) and at the end of treatment (macroscopically / microscopically).

OTHER:
- Assessment of the slides: Dose selection for scoring for cytogenetic damage was based on the results of a previous check on slide and/or cell quality, number of analysable cells and nuclear fragmentation.
Evaluation criteria:
ACCEPTANCE CRITERIA
The in vitro micronucleus assay is considered valid if the following criteria are met:
- The quality of the slides allowed the evaluation of a sufficient number of analysable cells both in the control groups (vehicle/positive) and in at least three exposed test groups.
- Sufficient cell proliferation was demonstrated in the vehicle control.
- The number of cells containing micronuclei in the vehicle control was within the range of the laboratory’s historical negative control data (95 % control limit). Weak outliers can be judged acceptable if there is no evidence that the test system is not “under control”.
- The positive control substances both with and without S9 mix induced a distinct, statistically significant increase in the number of micronucleated cells in the expected range.

ASSESSMENT CRITERIA
A test substance is considered to be clearly positive if the following criteria are met:
• A statistically significant increase in the number of micronucleated cells was obtained.
• A dose-related increase in the number of cells containing micronuclei was observed.
• The number of micronucleated cells exceeded both the value of the concurrent vehicle control and the range of our laboratory’s historical negative control data (95% control limit)
A test substance is considered to be clearly negative if the following criterion is met:
• Neither a statistically significant nor dose-related increase in the number of cells containing micronuclei was observed under any experimental condition.
• The number of micronucleated cells in all treated test groups was close to the concurrent vehicle control value and within the range of our laboratory’s historical negative control data (95 % control limit).
Statistics:
The statistical evaluation of the data was carried out using an appropriate statistical analysis. The proportion of cells containing micronuclei was calculated for each test group. A comparison of the micronucleus rates of each test group with the concurrent vehicle control group was carried out for the hypothesis of equal proportions (i.e. one-sided Fisher's exact test).
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: Osmolality and pH values were not influenced by test substance treatment.
- Precipitation: Test substance precipitation in culture medium at the end of exposure period was observed in the absence of S9 mix at 80 μg/mL and above in the 1st Experiment and at 160 μg/mL in the 2nd Experiment. In addition, in the presence of metabolic activation precipitates in culture medium occurred at 10 μg/mL and above in both experiments (macroscopical assessment).

RANGE-FINDING/SCREENING STUDIES
The test substance was poorly soluble in all commonly used vehicles. Thus, test substance suspensions in culture medium were used in the pretest. Precipitation in culture medium was observed from the lowest applied concentration of 20.3 μg/mL onward 4 and 24 hours after start of exposure. No cytotoxicity was observed in the pretest when tested up to the highest required concentration of 2600 μg/mL. Based on the experience on solubility of the test substance in other in vitro test systems (internal data) the vehicle DMSO was used in both main experiments. However, precipitates in culture medium were still observed in both experiments. Therefore, in the main experiments of this micronucleus study concentrations at the border of solubility in culture medium were tested for cytogenetic damage.

GENOTOXICITY - MICRONUCLEUS ANALYSIS
In this study, no biologically relevant increase in the number of micronucleated cells was observed either without S9 mix or after the addition of a metabolizing system. In both experiments in the absence and presence of metabolic activation after 4 and 24 hours treatment with the test substance the values (0.3 – 0.9 % micronucleated cells) were close to the concurrent vehicle/negative control values (0.3 - 0.8 % micronucleated cells) and within the range of the 95 % control limit of our historical negative control data (0.0 - 1.0 % micronucleated cells).
Besides, in the 1st Experiment in the absence of S9 mix the micronucleus rates were concentration-related increased at 40, 80 and 160 μg/mL (0.5 %, 0.6 % and 0.9 % micronucleated cells, respectively). However, all values were clearly within the range of the 95 % control limit of our historical negative control data range and, therefore, this finding has to be regarded as biologically irrelevant.
The positive control substances ethyl methanesulfonate and cyclophosphamide induced statistically significant increased micronucleus frequencies in both independently performed experiments in at least one positive control group each. In this study, in the absence and presence of metabolic activation the frequency of micronucleated cells (1.6– 5.9 % micronucleated cells) was above the range of our historical negative control data (0.1 - 1.5 % micronucleated cells) and close to or within our historical positive control data range (2.3 – 13.8 % micronucleated cells). Unfortunately, in the 1st Experiment in the absence of S9 mix the positive control EMS did not show the expected increase at 500 μg/mL (1.3 % micronucleated cells). But the second positive control group of 600 μg/mL EMS led to the expected statistically significant increase of the micronucleus rate (2.3 % micronucleated cells).

CYTOTOXICITY - RELATIVE POPULATION DOUBLING
In both main experiments in the absence and presence of S9 mix no cytotoxicity indicated by reduced RPD of below 50 % of control was observed up to the highest applied test substance concentrations. These values were calculated based on cell numbers determined at the end of each experiment. In the pretest the parameter RPD is a valuable indicator of test substance toxicity. However, in the main experiments due to the use of the cytokinesis block method it is a measure of cell proliferation only until addition of cytochalasin B to the cultures. But, it also gives an useful information on cell loss due to test substance exposure.

CYTOTOXICITY - PROLIFERATION INDEX
No clearly reduced proliferative activity was observed either after 4 hours exposure interval in the absence and presence of S9 mix or after 24 hours continuous test substance treatment in the test groups scored for cytogenetic damage. However, slightly elevated cytostasis indicated by reduction of the CBPI was obtained in the absence of S9 mix at 160 μg/mL (25.4 % of control) in the 2nd Experiment.
Due to the use of DMSO either as vehicle for the test substance or as solvent for cytochalasin B a final concentration of 1.5 % (v/v) DMSO was reached in the treatment medium in the 2nd Experiment at 24 hours continuous treatment in the absence of S9 mix. Therefore, a negative control culture supplemented with cytochalasin B (0.5 % [v/v] DMSO) only was run in parallel. This culture showed only a slightly higher CBPI (absolute value: 2.19) than the vehicle control (absolute value: 2.12). Thus, based on these data it was confirmed that the applied DMSO concentration had no detrimental impact on the outcome of this experimental part.

CELL MORPHOLOGY
Cell attachment/morphology was adversely influenced (grade > 2) only in the 1st Experiment in the presence of metabolic activation at 160 μg/mL.

Table 2. Historical negative control data, Cytochalasin B Method, Period: December 2013 - December 2014

 

Without S9 mix - all vehicles*

With S9 mix - all vehicles*

 

Micronucleated Cells [%]

Micronucleated Cells [%]

Exposure / Sampling period

4 h / 24 h & 24 h / 24 h

4 h / 24 h & 4 h / 44 h

Mean

0.5

0.6

Minimum

0.2

0.1

Maximum

0.9

1.5

Standard Deviation

0.2

0.3

95% Lower Control Limit

0.1

0.0

95% Upper Control Limit

0.8

1.1

No. of Experiments

43

42

* = culture medium, DMSO 1% (v/v), acetone 1% (v/v), ethanol 1% (v/v)

 

Table 3. Historical positive control data, Cytochalasin B Method, Period: December 2013 - December 2014

 

Without S9 mix

Ethyl methanesulfonate (EMS) 300 - 500 μg/mL

With S9 mix

Cyclophosphamide (CPP) 0.5 - 1.0 μg/mL

 

Micronucleated Cells [%]

Micronucleated Cells [%]

Exposure / Sampling period

4 h / 24 h & 24 h / 24 h

4 h / 24 h & 4 h / 44 h

Mean

3.0

5.4

Minimum

2.3

2.6

Maximum

6.4

13.8

Standard Deviation

0.8

2.3

95% Lower Control Limit

1.4

0.7

95% Upper Control Limit

4.7

10.1

No. of Experiments

43

42

Conclusions:
Negative
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
other: read across from analogue substance
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
HIS/TRP
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
33 μg - 6300 μg/plate (SPT, all tester strains)
3.3 μg - 1000 μg/plate (Prival; TA 100, TA1537 and TA 98)
Vehicle / solvent:
- Vehicle used: DMSO
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
congo red
other: 2-aminoanthracene (2-AA), N-methyl-N'-nitro-N-nitrosoguanidine, 4-nitro-o-phenylenediamine
Remarks:
S9-mix from rats and hamsters was used
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation
secondo experiment with Prival method

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
Acceptance criteria
Generally, the experiment was considered valid if the following criteria were met:
• The number of revertant colonies in the negative controls was within the range of the
historical negative control data for each tester strain.
• The sterility controls revealed no indication of bacterial contamination.
• The positive control substances both with and without S9 mix induced a distinct increase in
the number of revertant colonies within the range of the historical positive control data or
above.
• Fresh bacterial culture containing approximately 10E9 cells per mL were used.

Assessment criteria
The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least
doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and
E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA
1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either
without S9 mix or after adding a metabolizing system.
A test substance was generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the historical negative control
range under all experimental conditions in at least two experiments carried out
independently of each other.
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
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:
valid
Positive controls validity:
valid

MUTAGENICITY

Standard plate test

Tests without S9 mix

TA 1535: No relevant increase in the number of his+ revertants.

TA 100: Increase in the number of his+ revertants at 333 and 1000 μg/plate (factors 2.1 and 2.1, respectively).

TA 1537: Increase in the number of his+ revertants at 333 and 1000 μg/plate (factors 3.7 and 4.3, respectively).

TA 98: Increase in the number of his+ revertants at 33, 100, 333, 1000 and 3150 μg/plate (factors 4.0, 7.8, 12.8, 18.7 and 23.8,

respectively).

E. coli WP2 uvrA: No relevant increase in the number of trp+ revertants.

Tests with S9 mix

TA 1535: No relevant increase in the number of his+ revertants.

TA 100: Increase in the number of his+ revertants at 100, 333 and 1000 μg/plate (factors 2.2, 2.5 and 2.3, respectively).

TA 1537: Slight increase in the number of his+ revertants at 333 μg/plate (Factor 2.9).

TA 98: Increase in the number of his+ revertants at 33, 100, 333, 1000 and 3150 μg/plate (factors 5.0, 7.3, 13.1, 13.7 and 17.1,

respectively).

E. coli WP2 uvrA: No relevant increase in the number of trp+ revertants.

Prival preincubation test

Tests without S9 mix

TA 100: Increase in the number of his+ revertants at 333 and 1000 μg/plate (factors 1.9 and 2.6, respectively).

TA 1537: Increase in the number of his+ revertants at 333 and 1000 μg/plate (factors 6.0 and 7.8, respectively).

TA 98: Increase in the number of his+ revertants at 10, 33, 100, 333 and 1000 μg/plate (factors 3.3, 5.0, 8.7, 12.5 and 33.6,

respectively).

Tests with S9 mix

TA 100: Increase in the number of his+ revertants at 333 and 1000 μg/plate (factors 2.1 and 2.1, respectively).

TA 1537: Increase in the number of his+ revertants at 333 and 1000 μg/plate (factors 4.0 and 4.1, respectively).

TA 98: Increase in the number of his+ revertants at 33, 100, 333 and 1000 μg/plate (factors 2.1, 4.3, 8.9 and 14.5, respectively).

TOXICITY

A bacteriotoxic effect (decrease in the number of his+ or trp+ revertants) was observed in the standard plate test depending on the strain and test conditions from about 1000 μg/plate onward.

In the prival preincubation assay bacteriotoxicity (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants) was not observed up to the highest required concentration.

SOLUBILITY

Test substance precipitation was found from 333 μg/plate onward with and without S9 mix.

DISCUSSION

According to the results of the present study the test substance led to an evident and dose dependent increase in the number of his+ revertants with the tester strains TA 100, TA 1537 and TA 98, all with and without S9 mix. The increase of revertants was reproducible in two experiments (standard plate and prival preincubation test) carried out independently of each other. Based on the recent assessment criteria the test substance has to be considered positive.

Besides, the results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study.

In this study with and without S9 mix, the number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.

In addition, the positive control substances both with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data or above.

CONCLUSION

Thus, under the experimental conditions chosen here, it is concluded that Eukesolar Black ER Liquid - dried is a potent mutagenic test substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
other: read across from analogue substance
Adequacy of study:
weight of evidence
Study period:
2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
nitroreductase-deficient strains TA98NR and TA100NR
Principles of method if other than guideline:
The objective of this study was to evaluate the ability of test item to induce reverse mutations in histidine-requiring strains of Salmonella typhimurium in the absence and presence of a reductive hamster liver metabolising system (S-9). By assessing the mutagenicity of test item in nitroreductase deficient strains (TA98NR and TA100NR) alongside parent nitroreductase competent strains (TA98 and TA100), the role of nitroreduction in any test article related mutagenic activity could be determined.
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
nitroreductase deficient
Remarks:
TA98NR and TA100NR
Metabolic activation:
with and without
Metabolic activation system:
The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was obtained from Molecular Toxicology Incorporated, USA where it was prepared from uninduced male Golden Syrian hamsters. The S-9 was stored frozen at <-50°C, and thawed prior to use. Each batch was checked by the manufacturer for sterility, protein content, ability to convert ethidium bromide and cyclophosphamide to bacterial mutagens, and cytochrome P 450-catalysed enzyme activities (alkoxyresorufin-O-dealkylase activities).
Treatments were carried out both in the absence and presence of S-9 by addition of either buffer solution or 30% reductive (Prival) S-9 mix respectively. The composition of the mix and buffer solution are described in the following table:

Ingredient Final concentration/ml in:
30% reductive S9 mix Bufer solution
Sodium phosphate buffer pH 7.4 (SPB) 100 μmoles 100 μmoles
Glucose-6-phosphate (disodium) (G-6-P) 20 μmoles -
NADP(disodium) 4 μmoles -
NADH 2 μmoles -
Flavin mononucleotide (FMN) 2 μmoles -
Glucose-6-phosphate dehydrogenase 30 units -
Magnesium chloride (MgCl2) 8 μmoles -
Potassium chloride (KCl) 33 μmoles -
Water To volume To volume
S-9 (uninduced hamster liver) 300 μL -
Test concentrations with justification for top dose:
Mutation experiment:
2.5, 8, 25, 80, 250, 800 and 2500 µg/plate in the presence of a modified (reductive) S 9 mix
0.125, 0.4, 1.25, 4, 12.5. 40, 125, 400 and 1250 µg/plate in the absence of S-9.


The maximum treatment concentrations were limited by solubility of the test article in the primary vehicle, DMSO, and in the absence of S-9 also by the maximum volume additions that could be employed due to apparent vehicle-related toxicity. Following these treatments, evidence of toxicity was observed in all strains in the absence and presence of S-9. These toxic effects were observed at the highest treatment concentration of 1250 µg/plate in all strains except TA102 in the absence of S-9, and in strain TA102 and all strains in the presence of S-9 the toxic effects extended down to differing concentrations in each case that were between 8 and 800 µg/plate. The most extensive toxicity was observed in strains TA1535 and TA1537 in the presence of S-9, where only one or two non-toxic treatment concentrations remained. As mutation data were therefore available from fewer than 5 analysable concentrations for these strain treatments, these were repeated to provide a more thorough and robust assessment of the mutagenicity of the test article in this assay system
Vehicle / solvent:
anydrous analytical grade DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
congo red
mitomycin C
other: Metronidazole, 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in a pre-incubation methodology
- the experiment is performed in triplicate

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 30 minutes
- Exposure duration/duration of treatment: 3 days, plasted inverted and protected from light


Treatments were performed using a pre-incubation methodology in the absence and presence of a modified (reductive) S-9 mix. These platings were achieved by the following sequence of additions to sterile pre incubation tubes:
• 0.1 mL of bacterial culture
• 0.1 mL of test article formulation/vehicle control or 0.05 mL of positive control
• 0.5 mL of 30% reductive S-9 mix or buffer solution.
Quantities of test article formulation or control solution, bacteria and S-9 mix or buffer solution detailed above, plus an additional 0.5 mL of 100 mM sodium phosphate buffer (pH 7.4), were mixed together and placed in an orbital incubator set to either 37C (for the treatments in the absence of S-9) or 30C (for treatments in the presence of S 9) for 30 minutes, before the addition of 2 mL of supplemented molten agar at 45±1°C followed by rapid mixing and pouring on to Vogel-Bonner E agar plates.
When set, the plates were inverted and incubated protected from light for 3 days in an incubator set to 37C. Following incubation, these plates were examined for evidence of toxicity to the background lawn, and where possible revertant colonies were counted

The addition of 0.5 mL of 100 mM sodium phosphate buffer (pH 7.4) to these Mutation Experiment treatments was employed to reduce the solvent concentration during the pre-incubation period. DMSO, and some other organic solvents, are known to be near to toxic levels when added at volumes of 0.1 mL in this assay system when employing the pre-incubation methodology. By employing the modification indicated, the DMSO concentration in the pre-incubation mix was decreased, in an attempt to minimise or eliminate any toxic effects of the solvent that may have otherwise occurred. In order to ‘correct’ for the additional volume in the pre-incubation mix, these were plated out using 2 mL of 1.125% top agar (rather than the 0.9% top agar used for ‘standard’ pre incubation treatments), therefore the additions to each plate were comparable to that of the other pre-incubation treatments.

It should be noted that data from the initial treatments of all the tester strains in the absence and presence of S-9 were invalidated, as the plates demonstrated evidence of vehicle-related toxicity, and toxic effects extended over almost all treatment concentrations (considered to have been exacerbated by the vehicle-related toxicity). Repeat treatments were performed (using the treatment methodology described above) with all strains in the absence and presence of S-9. The data from these repeat treatments in the presence of S-9 were considered acceptable (and are those presented as the Mutation Experiment data for each strain in the presence of S-9), but the data from the test article treatments in the absence of S-9 were again invalidated, due to toxic effects extending over almost all treatment concentrations, and evidence of vehicle-related toxicity.
Further repeat treatments of all strains in the absence of S-9 were therefore performed. The methodology was amended slightly in order to address the apparent vehicle-related toxicity. Vehicle and test article treatments were performed using volume additions reduced to 0.05 mL, and the further 0.5 mL addition of 100 mM sodium phosphate buffer (pH 7.4) was therefore not required, and consequently following the pre-incubation period, these treatments were mixed with 2 mL of supplemented 0.9% molten (top) agar and poured onto Vogel-Bonner E agar plates. These plates were then incubated and scored as indicated above.
Due to data from fewer than 5 analysable concentrations being available from the first repeat treatments of strains TA1535 and TA1537 in the presence of S-9, further treatments of these strains were also performed alongside the further repeat treatments in the absence of S-9, and employed the same modified treatment methodology (0.05 mL volume additions, no additional 100 mM sodium phosphate buffer (pH 7.4) added and plate out using 0.9% molten top agar). The results from these treatments are reported as the Mutation Experiment further treatments data.


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Revertant colonies were counted electronically using a Sorcerer Colony Counter (Perceptive Instruments) or manually where confounding factors such as intensely coloured agar or bubbles or splits in the agar affected the accuracy of the automated counter.

- OTHER:
Rationale for test conditions:
The objective of this study was to evaluate the ability of test item to induce reverse mutations in histidine-requiring strains of Salmonella typhimurium in the absence and presence of a reductive hamster liver metabolising system (S-9). By assessing the mutagenicity of test item in nitroreductase deficient strains (TA98NR and TA100NR, the tow commercially available at the time of testing) alongside parent nitroreductase competent strains (TA98 and TA100), the role of nitroreduction in any test article related mutagenic activity could be determined. As the test substance is an azo compound, testing in the presence of S-9 in this study was performed using a modified reductive (Prival) S-9 pre-incubation methodology, as it is known that azo compounds can be reduced to free aromatic amines, which can be mutagenic.
Evaluation criteria:
For valid data, the test article was considered to be mutagenic if:
1. A concentration related increase in revertant numbers was ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98, TA98NR, TA100 or TA100NR) or ≥3-fold (in strains TA1535 or TA1537) the concurrent vehicle control values
The test article was considered positive in this assay if the above criterion was met.
The test article was considered negative in this assay if the above criterion was not met.
Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Biological relevance was taken into account, for example consistency of response within and between concentrations.
Data from strain TA98 were compared (non-statistically) with that from TA98NR, and data from strain TA100 were compared with that from TA100NR. Where a mutagenic response was seen in one or both parent strains but was absent or much reduced in the corresponding NR variant strain(s), this was considered to be indicative that bacterial nitroreduction enzymes play a significant role in the mutagenicity of the test compound as observed in this study.
Statistics:
Individual plate counts were recorded separately and the mean and standard deviation of the plate counts for each treatment were determined. Control counts were compared with the laboratory’s historical control ranges (see Attachments).
The presence or otherwise of a concentration response was checked by non-statistical analysis, up to limiting levels (for example cytotoxicity, precipitation or 5000 μg/plate). However, adequate interpretation of biological relevance was of
critical importance.
Species / strain:
bacteria, other: TA 98NR
Metabolic activation:
without
Genotoxicity:
negative
Remarks:
except at the highet tested dose of 1250 ug/plate
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
bacteria, other: TA100 NR
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
bacteria, other: TA100 NR
Metabolic activation:
with
Genotoxicity:
negative
Remarks:
except at the highest tested dose of 2500 ug/plate
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Following test item treatments of all the test strains in the absence and presence of S-9, clear and concentration-related (in some cases up to the toxicity and/or precipitating range) increases in revertant numbers were observed in strains TA98 and TA98NR in the absence and presence of S-9, in strain TA1537 in the absence of S-9, and in strains TA100, TA100NR and TA102 in the presence of S-9 In each case, these increases were ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98, TA98NR, TA100 or TA100NR) or ≥3-fold (in strain TA1537) the concurrent vehicle control values, although it should be noted that in strains TA100 and TA100NR in the presence of S-9, the 2-fold threshold level was only achieved at the maximum treatment concentration of 2500 µg/plate

for the tables of the number of revertants per strain with and without metabolic, refer to the attached background information

Conclusions:
The substance was tested for gene mutation in bacteria following OECD 471 with nitroreductase deficinet strains. Under the experimental conditions the mutagenicity of the substance was reduced for the NR strains confirming that direct acting mutagenicity of the substances, based on the nitroreductase activity, plays a major role in the mutagenic properties of the substance
Executive summary:

The test substance was assayed for mutation in seven histidine-requiring strains (TA98, TA100, TA1535, TA1537, TA102, TA98NR and TA100NR) of Salmonella typhimurium, both in the absence and in the presence of a reductive hamster liver metabolising system (S-9) in a single experiment.

All test item treatments in this study were performed using formulations prepared in anhydrous analytical grade dimethyl sulphoxide (DMSO). As the test item is an azo compound, testing in the presence of S-9 in this study was performed using a modified reductive (Prival) S-9 pre-incubation methodology, as it is known that azo compounds can be reduced to free aromatic amines, which can be mutagenic.

Mutation Experiment treatments of the tester strains were performed using a pre‑incubation methodology using final concentrations of test substance at 2.5, 8, 25, 80, 250, 800 and 2500 µg/plate in the presence of a modified (reductive) S‑9 mix or at 0.125, 0.4, 1.25, 4, 12.5. 40, 125, 400 and 1250 µg/plate in the absence of S-9. The maximum treatment concentrations were limited by solubility of the test article in the primary vehicle, DMSO, and in the absence of S-9 also by the maximum volume additions that could be employed due to apparent vehicle-related toxicity. Following these treatments, evidence of toxicity was observed in all strains in the absence and presence of S-9. These toxic effects were observed at the highest treatment concentration of 1250 µg/plate in all strains except TA102 in the absence of S-9, and in strain TA102 and all strains in the presence of S-9 the toxic effects extended down to differing concentrations in each case that were between 8 and 800 µg/plate. The most extensive toxicity was observed in strains TA1535 and TA1537 in the presence of S-9, where only one or two non-toxic treatment concentrations remained. As mutation data were therefore available from fewer than 5 analysable concentrations for these strain treatments, these were repeated to provide a more thorough and robust assessment of the mutagenicity of the test article in this assay system.

The Mutation Experiment further treatments of strains TA1535 and TA1537 in the presence of a modified (reductive) S‑9 mix were performed using final concentrations of the test item at 0.125, 0.4, 1.25, 4, 12.5, 40, 125, 400 and 1250 µg/plate. Although the treatment concentrations were not markedly reduced compared to those previously described, a slightly modified methodology was employed (as was used for the treatments used to provide the Mutation Experiment data for all strains in the absence of S-9), whereby the test article and vehicle volume additions were reduced, as it was considered that a vehicle-related effect was exacerbating the observed test article-related toxicity. Following these treatments, no clear evidence of toxicity was observed.

Precipitation of test article was observed on all of the test plates treated at 250 µg/plate and above in each experiment.

Vehicle and positive control treatments were included for all strains in each experiment. The mean numbers of revertant colonies fell withinacceptable ranges for vehicle control treatments, and were elevated by positive control treatments.

Following test item treatments of all the test strains in the absence and presence of S-9, clear and concentration-related (in some cases up to the toxicity and/or precipitating range) increases in revertant numbers were observed in strains TA98 and TA98NR in the absence and presence of S-9, in strain TA1537 in the absence of S-9, and in strains TA100, TA100NR and TA102 in the presence of S-9. In each case, these increases were ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98, TA98NR, TA100 or TA100NR) or ≥3-fold (in strain TA1537) the concurrent vehicle control values, although it should be noted that in strains TA100 and TA100NR in the presence of S-9, the 2-fold threshold level was only achieved at the maximum treatment concentration of 2500 µg/plate.

The mutagenic response in the nitroreductase deficient strain TA98NR in the absence of S-9 was reduced in magnitude compared to that with the corresponding treatments in the parent (nitroreductase proficent) strain TA98. Comparison of the magnitude of responses in these two strains in the presence of S-9 suggested that they were comparable, when assessed in terms of fold increase compared to the concurrent control level. However, the vehicle control counts in strain TA98NR were much smaller (mean of 19.3 revertants/plate) compared to that in strain TA98 (mean of 53.3 revertants/plate). Although the vehicle counts in each strain were considered acceptable, the much smaller vehicle control counts in strain TA98NR served to exaggerate the relativeAcid Black 63:2response when assessed in terms of fold increase. When the mean number of induced revertants/plate were assessed, the largest increase in strain TA98 was 176 revertants/plate (229.3 – 53.3), as compared to the largest increase in strain TA98NR of just 79 revertants/plate (98.3 – 19.3), which indicated a reduced mutagenic response in the nitroreductase deficient strain TA98NR. When the mutagenicity in strains TA100 and TA100NR were compared, there were similar mutagenic responses observed in both strains in the presence of S‑9, but in the absence of S-9 there was no response seen in strain TA100NR compared to a weak mutagenic response observed in strain TA100. Although less clear than the differential between strains TA98 and TA98NR, and only apparent for the treatments in the absence of S-9, these data indicated a reduced mutagenic response in the nitroreductase deficient strain TA100NR. Overall, the data from strains TA98, TA98NR, TA100 and TA100NR were considered to indicate that nitroreduction does play a significant role in the mutagenic activity of the test item seen in this study.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

OECD 489, testing proposal, analogue substance, to be performed

Endpoint conclusion
Endpoint conclusion:
no study available (further information necessary)

Additional information

Justification for classification or non-classification

A read across approach is proposed for assessing the in vivo gene mutation properties of the taregt substance, by using the results of a Comet Assay OECD 489 submitted as testing proposal on analogue substance.

The Comet Assay requirement is the results of the application of Column 2 Annex VII of the REACH Regulation which requires that substances with positive results for in vitro gene mutation study in bacteria are further investigated for their mutagenic properties in vivo.

Based on the available information no final conclusion on genotoxicity classification can be drawn since further in vivo data are required.

The dossier will be promptly updated when reliable and pertinent results for the substance will be available.