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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:
2012-06-06 to 2012-09-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Principles of method if other than guideline:
first experiment 4 hours treatment with and without metabolic activation
second experiment 24 hours treatment without metabolic activation, 4 hours treatment with metabolic activation
GLP compliance:
yes (incl. certificate)
Type of assay:
other: mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
- Name of test material (as cited in study report): Glycerides, C16-18 mono-, di and tri-, hydrogenated, citrates, potassium salts
- Molecular weight: Not applicable (mixture)
- Substance type: Industrial Chemical
- Physical state: Pale yellow solid block
- Analytical purity: 100%
- Lot/batch No.: 105618
- Expiration date of the lot/batch: May 11, 2014
- Storage condition of test material: Room temperature

Method

Target gene:
HPRT
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/Beta-Naphtoflavone induced Rat liver S9
Test concentrations with justification for top dose:
Experiment I:
without metabolic activation: 4.7; 9.4; 18.8; 37.5; 75.0; 150.0 µg/mL
with metabolic activation: 4.7; 9.4; 18.8; 37.5; 75.0; 150.0 µg/mL
Experiment II:
without metabolic activation: 4.7; 9.4; 18.8; 37.5; 75.0; 150.0 µg/mL
with metabolic activation: 4.7; 9.4; 18.8; 37.5; 75.0; 150.0 µg/mL
The cultures at the lowest concentration of 4.7 µg/mL in the presence and absence of metabolic activation (experiment I and II) were not continued since a minimum of only four analysable concentrations is required by the guidelines.
Vehicle / solvent:
- Solvent used: Tetrahydrofuran (THF) (99.9%)
- Justification for choice of solvent/vehicle: Solubility properties
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Experiment I: 4 hours with and without metabolic activation, Experiment II: 24 hours without metabolic activation, 4 hours with metabolic activation
- Expression time (cells in growth medium): 72 hours
- Selection time (if incubation with a selection agent): 10 days

SELECTION AGENT (mutation assays): 6-Thioguanine

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: >1,5x10exp. 6

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered to be non-mutagenic in this system.
A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces reproducibly with one of the concentrations a mutation frequency that is three times higher than the spontaneous mutation frequency in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
In a case by case evaluation this decision depends on the level of the corresponding solvent control data.
Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
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 examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not effected pH 7.32 in the solvent control versus pH 7.31 at 1200 µg test item/mL
- Effects of osmolality: Not increased (357 mOsm in the solvent control versus 342 mOsm at 1200 µg test item/mL
- Precipitation: In the first experiment precipitation of the test item at the end of treatment was noted at 75 and 150 µg/mL with and without metabolic activation. In the second experiment precipitation as described above occurred at 75 and 150 µg/mL with and at 37.5 µg/mL and above without metabolic activation.

- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES:
The highest concentration used in the pre-test was 1200 µg/mL limited by the solubility of the test item in THF and aqueous medium. Test item concentrations between 9.4 µg/mL and 1200 µg/mL were used to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24 hours treatment) of metabolic activation. Relevant cytotoxic effects indicated by a relative suspension growth below 50 were noted at 1200 µg/mL following 24 hours treatment without metabolic activation. The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal to the test item.
The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal to the test item. Precipitation occurred at 75.0 µg/mL and above in the presence (4 hours treatment) and absence (4 and 24 hours treatment) of metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA: Complies

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No relevant toxic effects occurred up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment.

Any other information on results incl. tables

Summary Table
  relative relative relative mutant   relative relative relative mutant  
conc. P S9 cloning cell cloning colonies/ induction cloning cell cloning colonies/ induction
µg/mL mix efficiency I density efficiency II 106cells factor efficiency I density efficiency II 106cells factor
        % % %     % % %    
Column 1 2 3 4 5 6 7 8 9 10 11 12 13
Experiment I / 4 h treatment       culture I          culture II
Solvent control with THF - 100.0 100.0 100.0 12.3 1.0 100.0 100.0 100.0 13.8 1.0
Positive control (EMS) 150.0 - 84.1 132.6 113.8 70.9 5.8 80.4 76.7 63.8 114.8 8.3
Test item 4.7 - 97.9 culture was not continued# 87.8 culture was not continued#
Test item 9.4 - 84.6 104.9 118.8 13.4 1.1 79.6 106.1 55.7 24.3 1.8
Test item 18.8 - 88.6 142.2 100.7 9.8 0.8 98.8 105.7 31.0 47.0 3.4
Test item 37.5 - 80.4 155.1 139.6 12.3 1.0 83.9 76.0 39.0 25.3 1.8
Test item 75.0 P - 68.5 70.8 113.2 8.3 0.7 82.0 96.4 42.7 25.1 1.8
Test item 150.0 P - 80.7 90.3 126.2 7.4 0.6 69.8 80.6 35.6 84.9 6.1
Experiment I / 4 h treatment       culture I          culture II
Solvent control with THF + 100.0 100.0 100.0 11.9 1.0 100.0 100.0 100.0 10.7 1.0
Positive control (DMBA) 1.1 + 58.3 70.4 70.2 802.4 67.3 85.0 48.2 119.1 274.5 25.5
Test item 4.7 + 81.4 culture was not continued# 113.4 culture was not continued#
Test item 9.4 + 77.8 120.4 92.1 28.2 2.4 96.0 60.9 109.9 13.3 1.2
Test item 18.8 + 80.5 105.1 100.7 10.3 0.9 119.3 87.5 92.3 17.5 1.6
Test item 37.5 + 78.5 130.0 100.3 11.5 1.0 119.0 97.9 105.2 8.5 0.8
Test item 75.0 P + 72.4 100.9 119.0 9.5 0.8 128.4 77.6 109.0 7.4 0.7
Test item 150.0 P + 72.6 98.2 107.7 6.7 0.6 131.9 76.2 121.7 15.1 1.4
Experiment II / 24 h treatment       culture I          culture II
Solvent control    - 100.0 100.0 100.0 20.4 1.0 100.0 100.0 100.0 9.7 1.0
Positive control (EMS) 150.0 - 108.1 79.6 86.7 381.1 18.7 105.1 126.4 103.5 422.4 43.7
Test item 4.7 - 109.1 culture was not continued# 106.8 culture was not continued#
Test item 9.4 - 98.6 108.0 84.6 26.2 1.3 105.6 95.6 117.2 23.1 2.4
Test item 18.8 - 98.8 92.3 92.4 34.1 1.7 109.3 75.7 98.4 20.2 2.1
Test item 37.5 P - 102.2 93.8 85.5 14.7 0.7 106.1 93.8 99.1 9.9 1.0
Test item 75.0 P - 97.2 104.0 84.7 24.0 1.2 105.8 144.5 113.3 13.2 1.4
Test item 150.0 P - 99.0 115.4 79.0 30.3 1.5 103.9 94.1 95.9 2.6 0.3
Experiment II / 4 h treatment          
Solvent control with THF   + 100.0 100.0 100.0 35.2 1.0 100.0 100.0 100.0 21.0 1.0
Positive control (DMBA) 1.1 + 103.0 120.7 67.8 732.9 20.8 100.0 105.0 93.9 348.7 16.6
Test item 4.7 + 104.4 culture was not continued# 101.6 culture was not continued#
Test item 9.4 + 123.9 109.0 98.6 14.8 0.4 108.7 107.6 95.2 4.5 0.2
Test item 18.8 + 133.2 89.6 93.2 29.6 0.8 112.2 109.0 98.7 10.8 0.5
Test item 37.5 + 94.0 127.6 79.8 26.9 0.8 100.9 93.4 101.3 16.4 0.8
Test item 75.0 P + 97.8 95.3 74.5 23.4 0.7 105.7 123.3 107.4 15.7 0.7
Test item 150.0 P + 106.3 104.3 74.7 15.7 0.4 105.3 99.5 94.7 14.2 0.7

#   culture was not continued since a minimum of only four analysable concentrations is required

P  precipitation observed at the end of treatment

Applicant's summary and conclusion