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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
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
May 28 to August 5, 2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: well documented study performed according to OECD Guideline and GLP. For read-across justification see Section 13.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): LINPLAST 810 TM unstab.
- Physical state: liquid

- Molecular formula (if other than submission substance): C35 H58 O6
- Molecular weight (if other than submission substance): 574.85
- Smiles notation (if other than submission substance): O=C(c1ccc(cc1C(=O)O)C(=O)OCCCCCCCC)OCCCCCCCCCC
- InChl (if other than submission substance): 1S/C27H42O6/c1-3-5-7-9-11-12-14-16-20-33-27(31)23-18-17-22(21-24 (23)25(28)29)26(30)32-19-15-13-10-8-6-4-2/h17-18,21H,3-16,19-20H2,1-2H3, (H,28,29)

Method

Target gene:
thymidine kinase locus
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media:
Minimal medium A: RPMI 1640 medium supplemented with penicillin, streptomycin sulphate, sodium pyrovate, L-glutamine, non-essential amino acids and F 68 Pluronic
Minimal medium B: same as Minimal medium A without F68 Pluronic
Complete medium (5%): Minimal medium A supplemented with 5% v/v heat-inactivated horse serum
Complete Medium (10%: Minimal medium A supplemented with 10% v/v heat-inactivated horse serum
Complete medium A (20%): Minimal medium A supplemented with 20% v/v heat-inactivated horse serum
Complete medium B (20%): Minimal medium B supplemented with 20% v/v heat-inactivated horse serum
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
other: Generation time and mutation rates (spontaneous and induced) were checked in the testing laboratory
Metabolic activation:
with and without
Metabolic activation system:
liver S9 mix of Phenobarbital - 5,6-Benzoflavone induced male Sprague Dawley rats
Test concentrations with justification for top dose:
With and without metabolic activation:
Toxicity test (range finding): 9.77, 19.5, 39.1, 78.1, 156, 313, 625, 1250 and 2500 µg/mL
1. experiment: 156, 313, 625 and 1250 and 2500 µg/mL with and without metabolic activation
2. experiment: 156, 313, 625, 1250 and 2500 µg/mL without metabolic activation; resp. 875, 1138, 1479, 1923 and 2500 µg/mL with metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: solvent giving the best solubility/dispersal characteristics
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: in the absence of metabolic activation (4 h/24h exposure): methyl methanesulphonate (MMS, 5.0/10.0 µg/ml); in the presence of metabolic activation: Benzo(a)pyrene (B(a)P, 2.0 µg/mL)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Experiment 1: 3 h (with and without metabolic activation)
Experiment 2: 24 h (without metabolic activation) and 3 h (with metabolic activation)
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 14 days (mutation selection assay)

SELECTION AGENT (mutation assays): trifluorothymidine (TFT), final concentration: 3.0 µg/mL in Complete medium B (20%)

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: mutation selection assay: 2000 per well; cloning efficiency assay: 1.6 cells per well (8 cells/mL)

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency (Cell concentrations were adjusted to 8 cells/mL and, for each dose level, 0.2 mL was plated into 96 microtitre wells, incubated for 7 days. Wells containing viable clones were identified by the eye using background illumination and then counted.)

OTHER EXAMINTATIONS
- Colony sizing: small and large type mutants, estimated for solvent and positive controls
- Observations of pH and osmolality: the treatment solutions were measured during performance of one the main experiments
Evaluation criteria:
Criteria for a positive result:
- the induced mutant frequency is higher than the global evaluation factor suggested for the microwell method (126 x 10E-6) at one or more doses
- there is a significant dose-relationship as indicated by the linear trend analysis
Results which only partially satisfy the above criteria will be dealt with on a case-by-case basis. Similarly, positive responses seen only at high levels of cytotoxicity will require careful interpretation when assessing their biological significance. Any increase in mutant frequency should lie outside the historical control range to have biological relevance.
Statistics:
Statistical analysis was performed according to UKEMS guidelines (Robinson W.D., 1990). The following methods were applied:
- Results of individual plates within a replicate treatment were checked for consistency by calculation of Chi-square.
- Heterogeneity factors were calculated for survival, viability and mutation. Values obtained should not exceed 10.8 times the current heterogeneity factor where 10.8 is the one-sided 0.1% level of the F-distribution with 1 and infinitive degrees of freedom.
- Overall consistency was evaluated by calculation of the ratio of the heterogeneity factors of the experiment to the current heterogeneity factor. This ratio should not exceed the one-sided 1% critical values from the F-distribution.
- The estimated heterogeneity factors of the experiment were combined with the current heterogeneity factor to define the updated estimated factors.
- Comparison of each treatment with the control: for each comparison, the ratio Di²/var(Di) was compared to the critical values for the one tailed Dunnett`s test.
- Test for linear trend: The evaluation of a linear trend in mutant frequency with the treatment dose was performed using weighted regression. The slope and its variance var(b) were calculated to form the test statistic b²/var(b), which was compared to tabulated critical values of Chi-square with 1 degree of freedom.

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
Precipitation in all tested 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: no
- Effects of osmolality: no
- Water solubility: not soluble
- Precipitation: yes, particles in suspension and opacity were observed by adding solution at 250 mg/mL in ethanol to RPMI complete medium (10%) in a ratio 1:100 in the solubility trial, on the basis of this result a concentration of 2500 µg/mL was selected as the highest dose level to be used in the test. Opacity, resp. slight opacity was observed at concentrations of 1250, 625 and 313 µg/mL.
- Other confounding effects: no


RANGE-FINDING/SCREENING STUDIES: No relevant toxicity was noted at any dose level tested using the short treatment time. In the absence of S9 metabolic activation, using the long treatment time, slight reduction of relative survival (RS) was noted at several concentrations without any dose relationship.


COMPARISON WITH HISTORICAL CONTROL DATA: The solvent and positive control mean mutant fractions were within the normal ranges experienced in the testing laboratory.

Any other information on results incl. tables

Table 1a: Toxicity test in the absence of S9 mix (3h exposure)
Concentration
µg/mL)
Cloning efficiency Relative Survival (%)
0 (Solvent control) 0.98 100
9.77 0.95 92
19.5 1.18 100
39.1 0.98 88
78.1 1.16 101
156 1.18 106
313 1.23 104
625 0.91 73
1250 1.05 94
2500 1.06 88
Table 1b: Toxicity test in the absence of S9 mix (24h exposure)
Concentration
(µg/mL)
Cloning efficiency Relative Survival (%)
0 (Solvent control) 1.14 100
9.77 0.87 96
19.5 0.72 81
39.1 0.75 78
78.1 0.74 77
156 0.84 79
313 0.76 60
625 1.00 82
1250 0.96 98
2500 0.76 95
Table 1c: Toxicity test in the presence of S9 mix (3h exposure)
Concentration
(µg/mL)
Cloning efficiency Relative Survival (%)
0 (Solvent control) 1.18 100
9.77 1.18 97
19.5 1.03 99
39.1 1.18 113
78.1 1.18 103
156 1.00 101
313 1.20 85
625 1.18 90
1250 1.20 92
2500 1.14 103
Table 2a: Mutation test in the absence of S9 Mix (3h exposure) (Summary of means of data) 1. Experiment 
Concentration
(µg/mL)
Relative Total Growth (%) Mutant fraction
(x 10-6)
Induced mutant fraction (x 10-6)
0 (Solvent control) 100 59.5 N/A
156* 92 62.8 3.36
313* 89 65.8 6.29
625* 75 60.7 1.19
1250* 83 57.5 -
2500* 83 54.3 -
MMS 10.0 (Positive Control) 63 351.0 291.5**
N/A: not applicable
* precipitation ** Induced mutant frequency > global evaluation factor (GEF = 126 x 10E-6)
Induced mutant fraction (IMF): Mutant fraction of treatment minus mutant fraction of solvent control
- : IMF <= 0
Table 2b: Mutation test in the presence of S9 Mix (3h exposure) (Summary of means of data) 1. Experiment
Concentration
(µg/mL)
Relative Total Growth (%) Mutant fraction
(x 10-6)
Induced mutant fraction (x 10-6)
0 (Solvent control) 100 52.2 N/A
156* 123 48.0 -
313* 94 64.5 12.30
625* 90 71.9 19.79
1250* 96 67.1 14.89
2500* 99 63.5 11.36
B(a)P 2.00 (Positive control) 55 580.5 528.4**
N/A: not applicable
* precipitation ** Induced mutant frequency > global evaluation factor (GEF = 126 x 10 E-6)
Induced mutant fraction (IMF): Mutant fraction of treatment minus mutant fraction of vehicle group
- : IMF <= 0
Table 2c: Mutation test in the absence of S9 Mix (24h exposure) (Summary of means of data) 2. Experiment
Concentration
(µg/mL)
Relative total growth (%) Mutant fraction
(x 10-6)
Induced mutant fraction (x 10-6)
0 (Solvent control) 100 77.2 N/A
156* 86 60.1 -
313* 82 73.8 -
625* 87 60.1 -
1250* 89 74.4 -
2500* 102 78.8 1.22
MMS 5.00 (Positive control) 85 734.8 657.6**
N/A: not applicable
* precipitation ** Induced mutation frequency > global evaluation factor (GEF = 126 x 10 E-6)
Induced mutant fraction (IMF): Mutant fraction of treatment minus mutant fraction of vehicle group
- : IMF <= 0
Table 2b: Mutation test in the presence of S9 Mix (4h exposure) (Summary of means of data) 2. Experiment
Concentration
(µg/mL)
Relative total growth (%) Mutant fraction
(x 10-6)
Induced mutant fraction (x 10-6)
0 (Solvent control) 100 56.3 N/A
875* 81 62.6 6.27
1138* 136 96.2** 39.87**
1479* 90 54.9 -
1923* 79 70.2 13.89
2500* 81 60.8 4.56
B(a)P 2.00 (Positive control) 33 658.6 602.4***
N/A: not applicable
* precipitation ** statistically significant at p<0.05 *** Induced mutant frequency > global evaluation factor (GEF = 126 x 10 E-6)
Induced mutant fraction (IMF): Mutant fraction of treatment minus mutant fraction of vehicle group
- : IMF <= 0

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

1,2,4-Benzenetricarboxylic acid, decyl octyl ester is not mutagenic in mouse lymphoma L5178Y cells, in either the absence or the presence of S9 mix, when tested in ethanol, under the reported experimental conditions.
Executive summary:

A structural analogue of the substance (1,2,4 -Benzenetricarboxylic acid, decyl octyl ester) was examined for mutagenic activity by assaying for the induction of 5 -trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells after in vitro treatment, in the absence and presence of S9 metabolic activation, using a fluctuation method. The study was designed to comply with the experimental methods indicated in: Test method B.17 "in vitro mammalian cell gene mutation test" described in Council Regulation (EC) No. 440/2008 and OECD Guideline for the testing of chemicals No. 476.

No relevant increases in mutant frequencies were observed following treatment with the test item, in the absence or presence of S9 metabolism and it was concluded that the substance does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.