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Toxicological information

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:
03 Feb - 10 Jun 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Version / remarks:
adopted in 2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted in 2008
GLP compliance:
yes (incl. QA statement)
Remarks:
Landesamt für Umwelt, Mainz, Germany
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Test material

1
Reference substance name:
Fatty acids, C18-unsaturated, 1,6 Hexanediol Diester
EC Number:
947-912-3
Molecular formula:
Not applicable for UVCB
IUPAC Name:
Fatty acids, C18-unsaturated, 1,6 Hexanediol Diester

Method

Target gene:
thymidine kinase locus (Tk1)
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: ATCC (Wesel, Germany)
- Suitability of cells: The cell line is characterized by a high sensitivity to chemical mutagens, by a high proliferation rate, a high cloning efficiency and a stable spontaneous mutant frequency.
- Normal cell cycle time: 10 - 12 h

For cell lines:
- Absence of Mycoplasma contamination: Before freezing, each batch was screened for mycoplasma contamination.
- Methods for maintenance in cell culture: Cells were thawed 6 days prior treatment and cultivated in cell culture flasks.
- Doubling time: 10 - 12 h
- Modal number of chromosomes: 40 ± 2
- Periodically checked for karyotype stability: yes
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- Type and composition of media:

Complete culture medium: RPMI 1640 medium supplemented with 10% heat inactivated horse serum, 1% penicillin/streptomycin (10,000 U/mL penicillin and 10 mg/mL streptomycin) and 2% 100 mM sodium pyruvate.

Viability medium: RPMI 1640 medium supplemented with 15% heat inactivated horse serum, 1% penicillin/streptomycin (10,000 U/mL penicillin and 10 mg/mL streptomycin) and 2% 100 mM sodium pyruvate.

Selection medium: RPMI 1640 medium supplemented with 15% heat inactivated horse serum, 1% penicillin/streptomycin (10,000 U/mL penicillin and 10 mg/mL streptomycin), 2% 100 mM sodium pyruvate and 5 µg/mL trifluorothymidine.

- Incubation (CO2 concentration, humidity level, temperature): The cells were cultured at 37.0 ± 1.0°C in a humidified atmosphere with 5.0 ± 0.5% CO2.
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system: Cofactor supplemented post-mitochondrial fraction (S9 mix)
- Source of S9 : Trinova Biochem GmbH, Gießen, Germany
- Method of preparation of S9 mix: S9 mix was prepared from the livers of male Sprague Dawley rats treated with an intraperitoneal injection of 500 mg/kg bw Aroclor 1254.
Test concentrations with justification for top dose:
Pre-test on toxicity
With and without S9 mix: 0.03, 0.06, 0.12, 0.24, 0.48, 0.95 and 1.907 mg/mL (4 h)

Experiment I
With and without S9 mix: 0.007, 0.015*, 0.030*, 0.060*, 0.119* and 0.238* mg/mL (4 h)

Experiment II
Without S9 mix: 0.003, 0.007, 0.015, 0.030*, 0.060*, 0.119* and 0.238* mg/mL (24 h)

* Concentrations evaluated for mutation rates
Vehicle / solvent:
- Vehicle used: acetone

- Justification for choice of vehicle: The solubility of the test item was determined in a non-GLP pre-test in culture medium (RPMI 1640) at a concentration of 19.07 mg/mL and in dimethyl sulfoxide (DMSO), ethanol as well as in acetone at a concentration of 381.4 mg/mL. The test item was completely insoluble in RPMI 1640, DMSO and ethanol, but soluable in acetone.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: Three experiments were performed, one Experiment I and two Experiment II. In the first Experiment II various acceptability criteria were not fulfilled, therefore the experiment was considered invalid and repeated. Only the valid experiment (second Experiment II) was included in the study report.

METHOD OF TREATMENT:
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 and 24 h
- Expression time (cells in growth medium between treatment and selection): 48 h
- Selection time: 10 - 12 days
- Method used: microtiter plates
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: After the expression period, 4000 cells/well from each experimental group were seeded into 2 microtiter plates with selective medium including trifluorothymidine (TFT). The viability (cloning efficiency 2) was determined by seeding 2 cells per well into 2 microtiter plates (same medium without TFT).
- Criteria for small (slow growing) and large (fast growing) colonies: Colonies were counted manually under a binocular magnifying glass. In accordance with their size, the colonies were classified into two groups:
Less than 25% of the well’s diameter = small colony
More than 25% of the well’s diameter = large colony

SELECTION AGENT: 5 μg/mL trifluorothymidine

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative total growth (RTG)

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- Method: Mutant frequency (MF)
Evaluation criteria:
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if:
• The induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 10E6 cells above the corresponding solvent control.
• The relative increase of the mutation frequency shows a dose relationship.

Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly negative if:
• The induced mutation frequency does not exceed a threshold of 126 colonies per 10E6 cells above the corresponding solvent control.
• The relative increase of the mutation frequency does not show a dose relationship.
Statistics:
A linear regression (least squares) of the test item concentrations was performed to assess a possible dose dependent increase of mutant frequencies. With the assessment of this regression, it can be evaluated whether mutations increase with increasing dose of the test item. A p-value of 0.05 or lower (significance level 95%) is considered as critical.
The positive controls were tested at one concentration only, therefore, no dose-dependency could be evaluated. The chi-square test was used.

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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: The pH values of solutions that were used in the preliminary cytotoxicity test were determined to exclude a negative influence on the assay. There was no critical change in pH value, therefore a negative influence on the assay was excluded.
- Data on osmolality: The osmolality of solutions that were used in the preliminary cytotoxicity test were determined to exclude a negative influence on the assay. There was no critical change in osmolality, therefore a negative influence on the assay was excluded.
- Precipitation and time of the determination: Precipitation (oily drops) of the test item was visible in both experiments at the maximum concentration of the test item (0.238 mg/mL).

RANGE-FINDING/SCREENING STUDIES: A pre-test was performed in order to determine the concentration range applicable for Experiment I and II. Seven test item concentrations in the range of 0.03 - 1.907 mg/mL were tested for 4 hours in the presence and absence of metabolic activation. Cytotoxicity (relative cloning efficiency) was determined for treated cells in comparison to controls. Precipitation was noted at 0.24 mg/mL and above with and without S9 mix. There was no cytotoxicity at any concentration, neither with nor without S9 mix.

STUDY RESULTS : There was no statistically significant or reproducible dose-dependent increase in the number of mutant colonies observed in any experiment at any of the tested concentrations, neither in the presence, nor in the absence of metabolic activation. No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentration of the test item. For details on gene mutation results and cytotoxicity in the main mutation experiments please refer to Table 2 under "Any other information on result incl. tables".

In total three experiments were performed (1x Experiment I and 2x Experiment II). The first Experiment II was invalid, since various acceptability criteria were not fulfilled. The invalid experiment was repeated and is reported as Experiment II in the study report. The invalid Experiment II is not included in the study report.

HISTORICAL CONTROL DATA: Please refer to Table 1 under "Any other information on materials and methods incl. tables".
- Positive historical control data: The mutation frequencies of both positive controls were within the range of the historical control data.
- Solvent historical control data: The mutation frequency of the solvent control acetone was outside the range of the historical control data. The historical control data for acetone only comprise two independent studies, therefore an untreated medium control was added to demonstrate that no deleterious or mutagenic effects were induced by acetone.
- Untreated negative historical control data: The mutation frequency of the untreated negative control was within the range of the historical control data.

Any other information on results incl. tables

Table 2: Experimental results

Content Relative Total Growth [%] Mutants per 106Cells
Exp I Exp II Exp I Exp II
4 h -S9 4 h +S9 24 h -S9 4 h -S9 4 h +S9 24 h -S9
Threshold - - - 207 190 250
Acetone - - - 81 64 124
Solvent pos. Control - - - 70 64 90
Positive control 60 35 33 365 491 575
0.003 mg/mL - - 105 - - n/e
0.007 mg/mL 103 83 91 n/e n/e n/e
0.015 mg/mL 111 83 90 76 83 n/e
0.030 mg/mL 102 89 103 86 82 103
0.060 mg/mL 108 86 100 87 71 106
0.119 mg/mL 105 96 100 83 62 113
0.238 mg/mL 101 66 108 71 77 101
Additional untreated control (medium control)
Solvent control (RPMI) - - - 85 64 90
Acetone 0.5% 90 109 98 81 64 124

n/e = not evaluated because the OECD 490 guideline requires only 4 concentrations

Applicant's summary and conclusion

Conclusions:
Under the conditions of the test the test item did not induce mutations in the Tk1 locus in the presence or absence of metabolic activation.