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Genetic toxicity: in vitro

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guidelineopen allclose all
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

Test material

Constituent 1
Reference substance name:
Reaction mass of sodium [1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and sodium bis[1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and sodium bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-)
IUPAC Name:
Reaction mass of sodium [1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and sodium bis[1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and sodium bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-)

Method

Target gene:
HPRT locus
Species / strain
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.
Controls
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.

Results and discussion

Test results
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).

Any other information on results incl. tables

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.

Applicant's summary and conclusion

Conclusions:
Negative with and without metabolic activation.