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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:
experimental study
Adequacy of study:
key study
Study period:
05 December 2017 - 01 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes
Type of assay:
other: in vitro mammalian cell gene mutation tests using the thymidine kinase gene (migrated information)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Physical appearance: pale yellow powder, free flowing crystalline
- Storage conditions: at room temperature desiccated

Method

Target gene:
Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells.
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: L5178Y/TK+/- -3.7.2C mouse lymphoma cells from American Type Culture Collection, (ATCC, Manassas, USA), (2001)
- Suitability of cells: recommended test system in international guidelines

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: 3 hr exposure: basic medium, supplemented with 5% (v/v) heat-inactivated horse serum (=R5 medium); 24 hr 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 (total amount of serum = 20%, R20-medium) and 5 µg/ml trifluorothymidine
* Non-selective medium: basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium)
- Properly maintained: yes, stock cultures of the cells were stored in liquid nitrogen (-196°C).
- Periodically checked for Mycoplasma contamination: yes
- Periodically 'cleansed' against high spontaneous background: yes, prior to testing
Metabolic activation:
with and without
Metabolic activation system:
Rat liver microsomal enzymes (S9 homogenate) from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg bw) and ß-naphthoflavone (100 mg/kg bw).
Test concentrations with justification for top dose:
Dose-range finding test (cytotoxicity test):
3 hour exposure (with and without S9): 63, 125, 250, 500, 1000 and 2000 µg/mL
24 hour exposure (without S9): 63, 125, 250, 500, 1000 and 2000 µg/mL

The dose levels for experiment 1 were based on the results of the dose-range finding study (precipitation of the test item was observed at 2000 µg/mL (3 hour treatment) and at and above 250 µg/mL (24 hour treatment).

Experiment 1
Dose levels used for the test:
* 3 hour exposure (without S9): 3.1, 6.3, 12.5, 25, 50, 100, 120, 140, 160, 180, 200, 225 and 250 µg/mL
* 3 hour exposure (with S9): 3.1, 6.3, 12.5, 25, 50, 75, 100, 120, 140, 160, 180, 200 and 250 µg/mL
Dose levels used to determine mutation frequencies:
* 3 hour exposure (without S9): 3.1, 6.3, 12.5, 25, 50, 100 and 120 µg/mL
* 3 hour exposure (with S9): 3.1, 6.3, 12.5, 50, 100, 140, 160 and 180 µg/mL

Experiment 2:
Dose levels used for the test:
* 24 hour exposure (without S9): 3.1, 6.3, 12.5, 25, 50, 75, 100, 120, 140, 160, 180, 200 and 250 µg/mL
Dose levels used to determine mutation frequencies:
* 24 hour exposure (without S9): 3.1, 12.5, 50, 75, 100, 120, 140 and 160 µg/mL
Vehicle / solvent:
- Solvent used: DMSO
- Justification for choice of solvent: a solubility test was performed based on visual assessment in an earlier study and the test item formed a clear colorless or (light) yellow solution in dimethyl sulfoxide (DMSO).
DMSO has been accepted and approved by authorities and international guidelines.
- The final concentration of the solvent in the exposure medium was 1% (v/v)
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): below 1 x 10^6 cells/mL for the 3 hour exposure period and 1.25 x 10^5 cells/mL for the 24 hour exposure period.

DURATION
- Cleansing period: 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.
- Exposure duration: 3 hours (experiment 1; with and without S9-mix); 24 hours (experiment 2; without S9-mix).
- Expression time: 2 days in which at least 4 x 10^6 cells were subcultured every day
- Selection time (if incubation with a selection agent): 11 or 12 days

ENVIRONMENTAL CONDITIONS (set to maintain):
All incubations were carried out in a humid atmosphere (80 - 100%, actual range 66 - 99%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 34.9 - 37.6°C).

SELECTION AGENT: TFT

STAIN: 0.5 mg/ml 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)

NUMBER OF REPLICATIONS:
- test concentrations: 1
- positive control: 1
- solvent control: 2

NUMBER OF CELLS EVALUATED: 9.6 x 10^5 cells per concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (relative suspension growth x relative cloning efficiency/100)
Evaluation criteria:
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 mutation frequency of more than the mutation frequency in the controls + 126 in a dose-dependent manner. An 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 the mutation frequency in the controls + 126.

ACCEPTABILITY CRITERIA:
a) The absolute cloning efficiency of the solvent controls is between 65 and 120% in order to have an acceptable number of surviving cells analyzed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is = 50 per 10^6 survivors and = 170 per 10^6 survivors.
c) The suspension growth over the 2-day expression period for the solvent controls should be between 8 and 32 for the 3 hour treatment, and between 32 and 180 for the 24 hour treatment.
d) The positive control should demonstrate an absolute increase in the total mutation frequency, that is, an increase above the spontaneous background MF (an induced MF (IMF)) of at least 300 x 10^-6. At least 40% of the IMF should be reflected in the small colony MF. And/or, the positive control has an increase in the small colony MF of at least 150 x 10^-6 above that seen in the concurrent solvent control (a small colony IMF of 150 x 10^-6).

Results and discussion

Test results
Key result
Species / strain:
mouse lymphoma L5178Y cells
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
- Precipitation: the test item precipitated in the exposure medium at concentrations of 1250 µg/mL and above. After 5 minutes, the test item precipitated in the exposure medium at the concentration of 2500 µg/mL.
- pH (at a concentration of 625 µg/mL): 7.22 (7.53 in the solvent control)
- Osmolarity (at a concentration of 625 µg/mL): 0.417 Osm/kg (0.438 Osm/kg in the solvent control)

RANGE-FINDING/SCREENING STUDIES:
- After 3 hours of treatment, in the absence of S9-mix, the relative suspension growth was 32% and 57% at the test item concentration of 125 µg/mL compared to the relative suspension growth of the solvent control, in the absence and presence of S9-mix, respectively. No cell survival was observed at test item concentrations of 250 µg/mL and above.
- After 3 hours of treatment, in the presence of S9-mix, the relative suspension growth was 101% at the test item concentration of 62.5 µg/mL compared to the relative suspension growth of the solvent control. No or hardly any cell survival was observed at test item concentrations of 125 µg/mL and above.
- After 24 hours of treatment, the relative suspension growth was 52% at the test item concentration of 125 µg/mL compared to the relative suspension growth of the solvent control. No cell survival was observed at test item concentrations of 250 µg/mL and above.

FIRST MUTAGENICITY TEST:
- Toxicity: in the absence of S9, the dose levels of 140-250 µg/mL were too toxic for further testing. In the presence of S9 the dose levels of 3.1-75 µg/mL showed no toxicity while dose levels of 200 and 250 µg/mL were too toxic for further testing.
- The dose level of 120 µg/mL was not used for mutation frequency measurement, due to a technical reason (rip in the culture bottle).
- In the absence of S9-mix, the relative total growth of the highest test item concentration was 13% compared to the total growth of the solvent controls.
- In the presence of S9-mix, the relative total growth of the highest test item concentration was 18% compared to the total growth of the solvent controls.
- No significant increase in the mutation frequency at the TK locus was observed after treatment with the test substance either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the test item treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

SECOND MUTAGENICITY TEST:
- Toxicity: the dose levels of 3.1-100 µg/mL showed no toxicity while dose levels of 180-250 µg/mL were considered too toxic for further testing.
- The relative total growth of the highest test item was 8% compared to the total growth of the solvent controls.
- No significant increase in the mutation frequency at the TK locus was observed after treatment with the test item. The numbers of small and large colonies in the test item treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

HISTORICAL CONTROL DATA: see table 1 and table 2

ACCEPTABILITY:
- 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 negative control database.
- Positive control chemicals both produced significant increases in the mutation frequency and the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database.
- The suspension growth over the two-day expression period for cultures treated with DMSO was between 16 and 22 (3 hour treatment) and 81 and 93 (24 hour treatment).

Applicant's summary and conclusion

Conclusions:
Based on the results of an in vitro mammalian cell gene mutation test, performed according to OECD guideline 490 and GLP principles, SHOP Ligand is not mutagenic in the TK mutation test system under the experimental conditions described in this report.