Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study is rated a "2" because it applied GLP, used appropriate testing procedures, but did not follow an accepted testing guideline.

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Results of the L5178Y Mouse Lymphoma Assay and the Balb/3T3 Cell In Vitro Transformation Assay for Eight Phthalate Esters
Author:
Barber ED, Cifone M, Rundell J, Przygoda R, Astill BD, Moran E, Mulholland A, Robinson E and Schneider B
Year:
2000
Bibliographic source:
J Appl Toxicol 20:69-80
Reference Type:
study report
Title:
Unnamed
Year:
1986
Report Date:
1986

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
L5178Y Mouse Lymphoma Assay and the Balb/3T3 Cell In Vitro Transformation Assay
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
Thymidine kinase (TK)
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Mouse lymphoma L5178Y cells in culture were used in the study. The cells were properly maintained in RPMI 1640 supplemented with F68 pluronic solution, L-glutamine, sodum pyruvate, and antibiotics, and horse serum (10% v/v) and periodically checked for mycoplasma contamination. In addition they were periodically "cleansed" against high spontaneous background.
Metabolic activation:
with and without
Metabolic activation system:
S9 derived from Aroclor 1254, rat liver
Test concentrations with justification for top dose:
The w/v concentrations of the doses assayed and density of test substance were not provided. The concentrations reported were in v/v. Six doses (2.0, 4.0, 5.0, 6.0, 8.0 and 10.0 ul/ml) were tested without S9 activation. Six doses (1.0, 2.5, 4.0, 5.0, 5.54 and 8.0 ul/ml) were tested with S9 activation. Mutant frequencies were determined for all evaluated levels.
Cytotoxic Concentration: 10 ul/ml
Vehicle / solvent:
Vehicle(s)/solvent(s) used: acetone
Justification for choice of solvent/vehicle: None provided
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
acetone
Positive controls:
yes
Remarks:
ethylmethanesulfonate, 3-methylcholanthrene
Details on test system and experimental conditions:
Mouse lymphoma cells were seeded into a series of tubes at 6 x 10E6 cells per tube. Dosed tubes were exposed for 4 hours to the test substance. An expression period of 48 hours was used; after the 48 hour expression time, 3 x 10E6 cells per plate were added to semi-solid selection medium containing 3 ug/ml 5-triflourothymidine (TFT) to score for mutant colonies and 200 cells per plate were added to cloning medium, without TFT, to evaluate viability. Mutant frequencies were calculated after 10-14 days incubation. Mutant and total colony counts at each dose level were determined by triplicate plates.

METHOD OF APPLICATION: Cloning medium consists of the RPMI 1640 growth medium with the addition of agar to a final concentration of 0.35% to achieve a semisolid state.

DURATION
- Preincubation period: 12 days
- Exposure duration: 4 hours
- Expression Time: 2 days
- Selection time (if incubation with a selection agent): 10-14 days

SELECTION AGENT (mutation assays): 5-trifluorothymidine

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: greater than 3 x 10E8 cells/ml

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
The assay was considered valid if all of the following criteria were satisfied:
1. The average cloning efficiency of the negative controls must be in the range of 60-130%.
2. The suspension growth, calculated as [(Day 1 cell count/3x10^5)] x [(Day 2 cell count)/(Day 1 split back volume density)], for the negative controls must be greater than or equal to 8.0.
3. The mutant frequency for the negative controls should be within the range of 10 x 10^-6 to 100x10^-6.
4. The mutant frequencies induced by the positive controls for either the nonactivation assay (using 0.3 ul/ml ethylmethanesulfonate) or the activation assay (using 2.5 ug/ml 3-methyl cholanthrene) must be greater than or equal to 200x10^-6.
5. For test materials determined to be non-mutagenic, the concentrations assayed must include one that will induce a reduction in the percent relative growth to 10-20% of the average for the negative controls, or a maximum concentration of 5ul/ml. The percent relative growth is an expression of toxicity calculated as the relative suspension growth x relative cloning efficiency/100.
6. The relative cloning efficiency must be greater than or equal to 10%; the total number of viable clones must be > 60 in order to accept an experimental mutant frequency.
7. A minimum of two dishes per set, with colony numbers that differ by no more than 3-fold, must be used to derive the mutant frequency.
8. A minimum of three test doses must be analyzed to evaluate the mutagenicity of the test substance.

In order to demonstrate mutagenesis for any given dose level, the mutant frequency must be greater than or equal to 150% the concurrent background mutant frequency plus 10x10^-6. To be considered a mutagen,
1. A toxicity-related or dose-related increase in the mutant frequencies must be demonstrated, or 2. if for a single dose near the max tested, mutant frequency is > 300% the concurrent background mutant frequency plus 20x10^-6.
Statistics:
The data were not evaluated for statistical significance.

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: Doses were selected to cover toxicity from little or no survival to 80-100% growth relative to the solvent control
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
pH: At the end of the 4 hour treatment period, the pH of the culture medium containing the highest concentration of test chemical was measured. All pH values were in the range of 7.21-7.47.
Remarks on result:
other: other: TK locus of mouse lymphoma cells, L5178Y cell line
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Cytotoxicity ranged from 3.5 - 21 % at the high dose levels. In the absence of activation, 2 to 10 ul/ml induced moderate to high toxicity (percent relative growths: 18.8% to 70.7%), but no increase in mutation frequency. In the presence of a metabolic fraction, 1 to 8 ul/ml was toxic (percent relative growths: 3.5% to 62.7%), without increasing the incidence of mutations. Thus, the test compound was considered non-mutagenic with activation in this assay.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under conditions of this study diundecyl phthalate was non-mutagenic in the mouse lymphoma assay with or without metabolic activation.
Executive summary:

In a mammalian cell gene mutation assay at the thymidine kinase locus, L5178Y mouse lymphoma cells cultured in vitro were exposed to the test substance, in acetone, at concentrations of 2.0, 4.0, 5.0, 6.0, 8.0, or 10.0 ul/ml in the absence of S9 mammalian metabolic activation or at concentrations of 1.0, 2.5, 4.0, 5.0, 5.54, or 8.0 ul/ml in the presence of S9.

The test substance was tested up to cytotoxic concentrations. Under non-activation conditions (dose levels 2 -10 ul/ml), the percent relative growth values were 70.7%-18.8%, indicating moderate to high toxicities, and the mutant frequencies were18.1 x 10 -6 to 30.7 x 10 -6. Because moderate to high toxicity was observed, but mutagenesis was not observed at any dose level assayed, the test substance was determined not to be a mutagen under non-activation conditions. In the S9 activation assay, the test substance was assayed at low to highly toxic dose levels (1 -8 ul/ml), and the mutant frequencies for the assayed dose levels were 21 x 10^-6 to 53.9 x 10^-6. The test substance was determined not to be a mutagen under S9 activation conditions. In both the non-activated and activated conditions, the positive controls induced the appropriate response. There was no evidence of induced mutant colonies over background as a result of exposure. The test material is considered inactive in the mouse lymphoma forward mutation assay with and without metabolic inactivation.