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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
The test substance, Formaldehyde, polymer with 1,3-benzenedimethanamine and phenol was accessed for its genotoxic ability in the Ames bacterial mutation assay, Chinese hamster ovary (CHO) cell in vitro chromosome aberration test and the L5178Y mouse lymphoma cell gene-mutation assay with colony sizing.
Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to the O.E.C.D. test guideline 476 with GLP compliance.
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
Target gene:
Thymidine kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
L5178Y cells, clone 3.7.2C, were obtained from Patricia Poorman-Allen, Glaxo Wellcome Inc., Research Triangle Park, NC Each lot of cryopreserved cells was tested using the agar culture and Hoechst staining procedures and found to be free of mycoplasma contamination. Prior to use in the assay, L5178Y cells were cleansed of spontaneous TK-/- cells by culturing in a restrictive medium (Clive and Spector, 1975).
Additional strain / cell type characteristics:
other: P53 gene defficient
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9 fraction.
Test concentrations with justification for top dose:
The concentrations chosen for cloning were 1.0, 2.0, 3.0, 4.0, and 5.0 µg/mL without S9 fraction activation and 5.0, 10, 25, and 50 µg/mL with S9 fraction metabolic activation.
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Methyl methanesulfonate at 15 and 20 ug/mL was used as the positive control for non-S9 fraction activated treatment. 7.12-Dimethylbenz(a)anthracene at concentrations of 1.25 and 1.5 ug/mL was used as the positive control for S9 fraction cultures.
Details on test system and experimental conditions:
The metabolic activation system was Aroclor 1254-induced rat liver S9 fraction was purchased by BioReliance from Moltox (Boone, NC) and stored at < 60oC until used. Immediately prior to use, the S9 fraction was mixed with the cofactors and Fischer's Medium for Leukemic Cells of Mice with 0.1% Pluronics (F0P) to contain 25 uL S9, 6.0 mg nicotinamide adenine dinucleotide phosphate (NADP), 11.25 mg DL-isocitric acid, and 975 uL F0P per mL S9-fraction activation mixture and kept on ice until used. The cofactor/F0P mixture was adjusted to pH 7.0 and filter-sterilized prior to the addition of S9.

The mutagenesis assay was performed in conical centrifuge tubes by combining 6 x 106 L5178Y/TK+/- cells, F0P medium (Fischer's Medium for Leukemic Cells of Mice with 0.1% Pluronics) or S9 activation mixture, and 100 uL dosing solution of test or control substance in solvent or solvent alone in a total volume of 10 mL. The positive controls were treated with MMS (at final concentrations in treatment medium of 15 and 20 ug/mL with a 4-hour exposure or 5.0 and 7.5 ug/ml with a 24-hour exposure) or 7,12 DMBA (at final concentrations in treatment medium of 1.5 and 1.25 ug/mL). Treatment tubes were gassed with 5±1% CO2 in air, capped tightly, and incubated with mechanical mixing for 4 or 24 hours at 37±1°C. The preparation and addition of the testsubstance dosing solutions were carried out under yellow lighting and the cells were incubated in the dark during the exposure period. After the treatment period, the cells were washed twice with F0P or F0P supplemented with 10% horse serum, 2 mM L-glutamine, 100 U penicillin/mL and 100 μg streptomycin/mL (F10P). After the second wash, the cells were resuspended in 20 mL F10P, gassed with 5±1% CO2 in air and placed on the roller drum apparatus at 37±1°C.

For expression of the mutant phenotype, TK-/- the cultures were counted using an electronic cell counter and adjusted to 3x105 cells/mL at approximately 24 and 48 hours after treatment in 20 and 10 mL total volume, respectively. For the 24-hour exposure, cultures were adjusted to 3x105 cells/mL in 20 mL immediately after test substance removal, then at 48 and 72 hours after treatment in 20 and 10 mL total volume, respectively. Cultures with less than 3x105 cells/mL were not adjusted.

For expression of the TK-/- cells, cells were placed in cloning medium (C.M.) containing 0.24% dissolved Noble agar in F0P plus 20% horse serum. Two flasks per culture to be cloned were labeled with the test substance concentration, activation condition, and either TFT (trifluorothymidine, the selective agent) or VC (viable count). Each flask was filled with 100 mL C.M. and placed in an incubator shaker at 37±1°C until used. The cells were centrifuged at 1000 rpm for 10 minutes and the supernatant was decanted. The cells were then diluted in C.M. to concentrations of 3x106 cells/100 mL C.M. for the TFT flasks and 600 cells/100 mL C.M. for the VC flasks. After the dilution, 1.0 mL of stock solution of TFT was added to the TFT flask (final concentration of 3 ug/mL) and both this flask and the VC flask were placed on the shaker at 125 rpm and 37±1°C. After 15 minutes, the flasks were removed and the cell suspension was dispensed equally into each of three appropriately labeled Petri dishes. To accelerate the gelling process, the plates were placed in cold storage (2-8°C) for approximately 30 minutes. The plates were then incubated at 37±1°C in a humidified 5±1% CO2 atmosphere for 10-14 days.

After the incubation period, the VC plates were counted for the total number of colonies per plate and the total relative growth determined. The TFT-resistant colonies were then counted for each culture with ≥ 10% total relative growth. The diameters of the TFT-resistant colonies for the positive and solvent controls and, in the case of a positive response, the test substance-treated cultures were determined over a range of approximately 0.2 to 1.1 mm. The rationale for this procedure is as follows: Mutant L5178Y TK-/- colonies exhibit a characteristic frequency distribution of colony sizes. Studies have demostrated that the small colony variants carry chromosome aberrations associated with chromosome 11, the chromosome on which the TK locus is located in the mouse.
Evaluation criteria:
The International Workshop on Genotoxicity established a Global Evaluation Factor (GEF) for a positive response in the mouse lymphoma gene-mutation test at an Induced Mutation Frequency (IMF) of ≥ 90 mutants per 106 clonable cells at the Aberdeen meeting in 2003, published in Moore et al., 2006.

A result was considered positive if a concentration-related increase in induced mutant frequency was observed in the treated cultures and one or more treatment conditions with 10% or greater total growth exhibited induced mutant frequencies of >90 mutants per 106 clonable cells.

A result was considered negative if the treated cultures exhibited induced mutant frequencies of less than 90 mutants per 106 clonable cells (based on the average mutant frequency of duplicate cultures) and there was no concentration-related increase in mutant frequency.
Statistics:
No data
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The test substance did not induce a significant increase of the TFT resitant mutant frequency without rat liver S9 metabolic activation. At the high concentration of 5 ug/mL without S9 metabolic activation and a 4 hr exposurse period the % Relative Total Growth was 5%. At the high concentration of 4 ug/mL without S9 metabolic activation and a 20 hr exposurse period the % Relative Total Growth was 23%. At the high concentration of 50 ug/mL with rat liver S9 metabolic activation and a 4 hr exposure period the % Relative Total Growth was 11%.

In the presence in of rat liver S9 metabolic activation and a 4 hr exposure the test substance induced a significant dose-related increase of the mean Induced Mutant Frequency of 92.5 at the high concentration of 50 ug/mL. The increase of the mutant frequency at 50 ug/mL was 2.65-fold the concurrent vehicle control mean mutant frequency value. Sizing of the TFT resistant mutants on the test substance S9 metabolic activation plates demonstrated an increase in the frequency of small colony mutants.
Remarks on result:
other: strain/cell type:
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
positive with metabolic activation

The data demonstrate that the test substance is mutagenic to mouse lymphoma cells when metabolically converted to an ultimate mutagen. The increase in the frequency of small colony mutants under the conditions of S9 metabolic activation suggests that the test substance is capable of causing chromosome damage in chromosome 11 of these mouse cells.
Executive summary:

The test substance, Formaldehyde, polymer with 1,3 -benzenedimethanamine and phenol was evaluated to induce gene-mutation in mouse lymphoma cells in culture in an O.E.C.D. test guideline 476 study. The test substance did not induce gene-mutation in the absence of rat liver fraction S9 metabolic activation. However, the data demonstrate that the test substance is mutagenic to mouse lymphoma cells when metabolically converted to an ultimate mutagen. The increase in the frequency of small colony mutants under the conditions of S9 metabolic activation suggests that the test substance is capable of causing chromosome damage in chromosome 11 of these mouse cells.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Additional information

Additional information from genetic toxicity in vitro:

The test substance did not induce evidence of genotoxicity in the Ames bacterial mutation and CHO in vitro chromosome aberration assays with or without rat liver derived S9 metabol;ic activation preparation when test upto cytotoxic concentrations.

The test substance induced a dose-related increase of the TFT resistant mutant frequency in L5178Y mouse lymphoma cells that reached a mean value of 92.5 induced mutants at the high concentration of 50 ug/mL. The mutant frequency observed at 50 ug/mL was 2.65 -fold the concurrent vehicle control value. An increase in the frequency of small colony mutants was observed suggesting that the test substance is capable of inducing chromosome aberration in chromosome 11 of the mouse lymphoma cells.


Justification for selection of genetic toxicity endpoint
Positive response observed for gene-mutation in L5178Y mouse lymphoma cells in the presence of rat liver S9 fraction metabolic activation.

Justification for classification or non-classification

Data from an in vivo genotoxicity test is currently not available for the test substance. Therefore, Classification and Labeling for potential gene-mutation can not be made at this time. An in vivo test for genotoxicity is planned.