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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:
06 July 2016 - 23 August 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2016

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 test

Test material

Reference
Name:
Unnamed
Test material form:
solid
Details on test material:
Appearance: Dark red-brown solid
Storage conditions: At room temperature
Specific details on test material used for the study:
- No correction factor for purity required.

Method

Target gene:
- Thymidine kinase locus
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
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 and ß-naphthoflavone.
Test concentrations with justification for top dose:
Dose-range finding test (with and without S9; cytotoxicity test): 0.85, 2.7, 8.5, 26 and 82 μg/mL
Since the test item precipitated directly in the exposure medium at concentrations of 52 μg/mL and above and after 10 minutes at 164 μg/mL and above, the highest concentration used in the dose range findinig test was 82 μg/mL.
Experiment 1 (with and without S9): 0.054, 0.17, 0.54, 1.7, 5.4, 17, 52 and 164 μg/mL. Doses were selected based on the results of the dose range finding test.
Experiment 2 (without S9): 0.054, 0.17, 0.54, 1.7, 5.4, 17 and 52 μg/mL. Doses were selected based on the results of the dose range finding test and experiment 1.
Vehicle / solvent:
- Vehicle used: ethanol
- Justification for choice of vehicle: as recommended in OECD test guideline 490
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Without S9
Untreated negative controls:
no
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

DURATION
- Exposure duration:
Short term treatment: 3 hours (with and without S9-mix)
Prolonged treatment: 24 hours (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

SELECTION AGENT: 5 µg/mL trifluorothymidine (TFT)

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 (see 'Any other information on materials and methods incl. tables' for details on calculations)
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 resultsopen allclose all
Key result
Species / strain:
mouse lymphoma L5178Y cells
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
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
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:
DOSE-RANGE FINDING STUDY
- Both in the absence and presence of S9-mix, no significant toxicity in the relative suspension growth was observed up to and including the highest test item concentration of 82 μg/mL compared to the suspension growth of the solvent control.
- No toxicity in the relative suspension growth was observed up to test item concentrations of 82 μg/mL compared to the solvent control.
- The test item precipitated in the exposure medium at the highest concentration tested (82 μg/mL) in the presence of S9-mix.

FIRST EXPERIMENT (Mutagenicity test)
- the test item precipitated at the highest concentration tested (164 μg/mL) in the presence of S9-mix.
- no significant toxicity was observed and all dose levels were evaluated in the absence and presence of S9-mix. In the absence of S9-mix, the relative total growth of the highest test item concentration was 48% compared to the total growth of the solvent controls. In the presence of S9-mix, no toxicity was observed up to and including the highest tested dose level tested
- No significant increase in the mutation frequency at the TK locus was observed either in the absence or in the presence of S9-mix. Increases above the 95% upper control limit were observed at the mid dose levels of 1.7 and 17 μg/ml in the presence of S9-mix. Although these increases are above the 95% upper control limit, no dose-related response is observed, and the increases in the mutation frequency (172 and 143 per 10^6 survivors, for 1.7 and 17 μg/ml, respectively) are below the MF(controls) + 126 (207 per 10^6 survivors), therefore these increases are considered to be not biologically relevant.

SECOND EXPERIMENT (Mutagenicity test)
- the test item precipitated at concentrations of 52 and 164 μg/mL after 24 hours in the absence of S9
- no toxicity was observed up to and including the highest dose level used for measurements (52 μg/mL)
- no significant increase in the mutation frequency at the TK locus was observed after treatment in the absence of S9-mix.

HISTORICAL DATA: see table 1 and table 2

ACCEPTABILITY CRITERIA
- The positive controls both produced significant increased in the mutation frequency, which was within the 95% control limits of the historical data range.
- The absolute cloning efficiency of the solvent controls (CEday2) was between 98 and 80 (without S9) and 76 and 65 (with S9) in experiment 1 and 83 and 91 in experiment 2.
- The spontaneous mutation frequency in the solvent control was 75 and 68 (without S9 and 76 and 86 (with S9) per 10^6 survivors in experiment 1 and 82 and 86 per 10^6 survivors in experiment 2. These frequencies were within the 95% control limits of the historical data range.
- The suspension growth over the two-day expression period for cultures treated with ethanol was between 10 and 25 (3 hour treatment) and 110 and 114 (24 hour treatment).
Remarks on result:
other: Experiment 1

Any other information on results incl. tables

For detailed results, see attached illustration.

Table 1 Historical Control Data of the Spontaneous Mutation Frequencies of the Solvent Controls for the Mouse Lymphoma Assay

 

Mutation frequency per 106 survivors

 

- S9-mix

+ S9-mix

 

3 hour treatment

24 hour treatment

3 hour treatment

Mean

83

75

84

SD

22

24

27

n

161

146

210

Upper control limit

(95% control limits)

128

126

141

Lower control limit

(95% control limits)

37

25

28

SD = Standard deviation

n = Number of observations

Table 2 Historical Control Data of the Mutation Frequencies of the Positive Controls for the Mouse Lymphoma Assay

 

Mutation frequency per 106 survivors

 

- S9-mix

+ S9-mix

 

3 hour treatment

24 hour treatment

3 hour treatment

Mean

894

724

1694

SD

247

200

793

n

81

74

108

Upper control limit

(95% control limits)

1431

1216

4045

Lower control limit

(95% control limits)

356

231

-657

SD = Standard deviation

n = Number of observations

Distribution historical solvent and positive control data from experiments performed between January 2013 and May 2016. 

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, X-19555 is not mutagenic in the TK mutation test system under the experimental conditions described in this report.