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Genetic toxicity: in vitro

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in vitro gene mutation study in mammalian cells
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Valid without restriction; the study was conducted in compliance with the Good Laboratory Practice regulations as set forth in the Organization for Economic Cooperation and Development Principles of Good Laboratory Practice C(81)30 (Final) Annex 2, issued 1979-1980 (effective 1981), with any applicable amendments and OECD Guideline 476 (In Vitro Mammalian Cell Mutation Assay).

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
Type of assay:
mammalian cell gene mutation assay

Test material

Details on test material:
- Material Identification: EC 95-0205, TXIB
- Lot number: 4008130
- Date received: May 18, 1995
- Physical Description: Clear, colorless liquid


Target gene:
Hypoxanthine-guanine phosphoribosyl transferase (HGPRT)
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
The CHO-K1-BH4 cells used in this study were obtained in October 1982 from Dr. A.W. Hsie (Oak Ridge National Laboratory, Oak Ridge, Tennessee). Master stocks of the cells were maintained frozen in liquid nitrogen. Laboratory cultures were maintained as monolayers at 37 °C ± 1.5 °C in a humidified atmosphere containing 5% ± 1.5% CO2. Laboratory cultures were periodically checked for karyotype stability and for the absence of mycoplasma contamination. To reduce the negative frequency of HGPRT mutants to as low a level as possible, the cell cultures were exposed to conditions which selected against the HGPRT phenotype. Cells were maintained in cleansing medium for 2-3 days, placed in recovery medium for 1 day, and then returned to culture medium. Cleansed cultures were used to initiate mutation assays from 3-7 days after having been removed from cleansing medium.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
The metabolic activation system was comprised of rat liver enzymes (S9 fraction; Arochlor 1254 treated) and an energy producing system, CORE (nicotinamide adenine dinucleotide phosphate, glucose-6-phosphate and an ion mix) prepared in a phosphate buffer.
Test concentrations with justification for top dose:
With and without metabolic activation:
3.89, 7.77, 15.5, 31.1, 62.2, 124, 249, 498, 995, 1990 µg/ml

Without metabolic activation:
10, 15, 20, 25, 30, and 40 µg/ml
With metabolic activation:
250, 500, 750, 1000, 1500, 2000 µg/ml
Vehicle / solvent:
Dimethyl sulphoxide (DMSO)
Controlsopen allclose all
Untreated negative controls:
Negative controls (media only) were performed by carrying cells unexposed to the test article through all of the assay operations. In the activation portion of the cytotoxicity assay, the negative controls were exposed to the S9 metabolic activation mix.
Negative solvent / vehicle controls:
Test article soluble in DMSO. Concurrent vehicle controls performed for each portion of assay by exposing cells to 1% DMSO in culture medium for 4 hours. In activation portion of assays, vehicle controls were also exposed to S9 metabolic activation mix.
True negative controls:
Positive controls:
Positive control substance:
other: 5-Bromo-2'-deoxyuridine (BrdU) was used at a concentration of 50 µg/ml as a concurrent positive control article for nonactivation mutation studies.
Triplicate cultures were used in the cytotoxicity assays and duplicate controls were used in the mutation assays. Negative controls were not used in the mutation assays.
Positive controls:
Positive control substance:
other: Methylcholanthrene (MCA) was used at 5 µg/ml as a concurrent positive control for mutation assays performed with S9 activation.
Details on test system and experimental conditions:
The cells used during experimental studies were maintained in Ham’s Nutrient Mixture F12 supplemented with L-glutamine, antibiotics, and fetal bovine serum (FBS; 8% by volume), also referred to as culture medium. Cleansing medium used to reduce spontaneous frequency of HGPRT mutants prior to experimental studies consisted of culture medium (5% serum) supplemented with thymidine, hypoxanthine, glycine, and either aminopterin or methotrexate. Recovery medium was similar to cleansing medium except the aminopterin or methotrexate component was removed and the FBS was increased to 8%. Selection medium for mutants was hypoxanthine-free F12 medium containing 4 μg/ml of 6-thioguanine (TG) and the fetal bovine serum component reduced to 5%.

Preparation of dosing solutions/ dosing procedure:
TXIB was dissolved in DMSO at 100X the highest desired treatment concentration. The primary stock solutions were then prepared by serial dilution with DMSO. Final 1X dosing stocks were prepared by making 1:100 dilutions of the primary stocks into culture medium containing 8% fetal calf serum for nonactivation studies and 5% serum for activation studies. Preparations of test article in vehicle were prepared fresh each day for testing. Treatments were initiated by replacing the culture medium on the cell cultures with the treatment medium containing the test article at the desired concentrations.

Ten triplicate concentrations were used that ranged from 3.89-1900µg/ml; higher concentrations were not included because of the insoluble nature of the test article. In addition, three negative (media) controls and three vehicle controls containing 1% DMSO were used in each cytotoxicity assay. The cells were seeded at 200 cells per dish, allowed to attach overnight and exposed to the test article or control article for 4 hours at 37°C ± 1.5°C in a humidified atmosphere containing 5% CO2. The cells were washed twice in Dulbecco’s phosphate buffered saline (PBS) and incubated in F12 culture medium for six additional days to allow colony development. Colonies were then fixed in alcohol, stained with Giemsa and counted by eye, excluding those with approximately 50 cells or less. Cytotoxicity was expressed as a percentage of colony counts in treated cultures versus control cultures. Data from this study were used to select doses for the mutation assay that covered a range from approximately 0% to 90% reduction in colony-forming ability.

Nonactivation Assay:

The cleansed cells were plated at 3X10^6 cells per tissue culture flask on the day before dosing. The time between plating and treatment was about 18 hours. Cell cultures were treated with test article in duplicate or control material in duplicate or triplicate for 4 hours at 37°C ± 1.5°C in a humidified atmosphere containing 5% CO2. After treatment, the cell monolayers were washed twice with phosphate buffered saline, trypsinized, and suspended in culture medium. The cell suspension from each dose level was counted using a Coulter Counter and replated at 1.5X10^6 cells into each of two 150-mm petri dishes and 200 cells into each of three 60-mm petri dishes. The small dishes were incubated for 7 days to permit colony development and the determination of cytotoxicity associated with each treatment. The large dishes were incubated for seven days to permit growth and expression of induced mutations. The mass cultures were subcultured every two to three days during the expression period to maintain logarithmic cell growth.

At the end of the expression period (7 days), each culture was reseeded at 2X10^5 cells per 100-mm dish (12 dishes total) in mutant selection medium. Also, three 60-mm dishes were seeded at 200 cells each in culture medium to determine the cloning efficiency of each culture. After incubation for 7-10 days at 37°C ± 1.5°C in a humidified atmosphere containing 5% CO2, the colonies were fixed with alcohol, stained with Giemsa and counted to determine the number of TG-resistant colonies in mutant selection dishes and the number of colonies in the cloning efficiency dishes.

The activation assay was performed independently with its own set of vehicle and positive controls. The procedure was identical to the nonactivation assay except for the addition of the S9 fraction of rat liver homogenate and necessary cofactors during the 4-hour treatment period. The fetal bovine serum content of the medium used for dosing was reduced to 5% by volume. The cofactors consisted of nicotinamide adenine dinucleotide phosphate (NADP, sodium salt), glucose-6-phosphate, calcium chloride, potassium chloride, and magnesium chloride, all of which were in a pH 7.8 sodium phosphate buffer.
Evaluation criteria:
The following results must be obtained to conclude that test material is a mutagen:
-A dose- or toxicity-related increase in mutant frequency should be observed, desirably for at least 3 doses.
-Significant mutagenic activity in one culture of a dose should be confirmed in replicate of same dose.
-A mutagenic dose-response in one mutation assay should be confirmed in second mutation assay.
-If an increase in mutant frequency is observed for a single dose near the highest testable toxicity (see above) and the number of mutant colonies is >2X that needed to indicate a significant response, test article generally will be considered mutagenic.
-For some test articles, correlation between toxicity and applied concentration is poor. Proportion of applied article that effectively interacts with cells to cause genetic alterations is not always repeatable or readily controlled. Conversely, measurable changes in frequency of induced mutants may occur with concentration changes that cause only small changes in observable toxicity. Therefore, either applied concentration or toxicity (percent survival) can be used to establish whether mutagenic activity is related to an increase in effective treatment.
-Treatments that reduce relative clonal survival to <5% may be included in assay but will not be sufficient evidence for mutagenicity as it relates to risk assessment.
A test article is evaluated as nonmutagenic in a single assay only if the minimum increase in mutant frequency is not observed for a range of applied concentrations that extends to concentrations causing 10 to 15% survival. If test article is relatively nontoxic, the maximum applied concentration will normally be 5 mg/ml or approximately twice the solubility limit in medium. If a repeat assay does not confirm an earlier, minimal response as discussed above, test article is considered nonmutagenic in this assay.

Assay Acceptance Criteria for the study is detailed below.
The statistical tables provided by Kastenbaum and Bowman (1970) are used to determine whether the results at each dose level are significantly different from the negative controls at 95% or 99% confidence levels. This test compares variables distributed according to Poissonian expectations by summing up the probabilities in the tails of two binomial distributions. The 95% confidence level must be met as one criterion for considering the test article to be active at a particular dose level. In addition, the mutant frequency must meet or exceed 15X10^-6 in order to compensate for random fluctuations in the 0 to 10X10^-6 background mutant frequencies that are typical for this assay.

Results and discussion

Test results
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
Vehicle controls validity:
Untreated negative controls validity:
Positive controls validity:
Additional information on results:
Under nonactivation conditions, the test article was excessively cytotoxic from 62.2-1990µg/ml and treatments at and below 31.1µg/ml were noncytotoxic. In the presence of metabolic activation, the test article was noncytotoxic from 3.89-1990µg/ml.

Two trials of the nonactivation assay were performed. In Trial 1, eleven duplicate treatments from 5.00-100µg/ml were initiated. The 5.00µg/ml treatment was terminated because sufficient weakly cytotoxic treatments were available for analysis. In addition, duplicate cultures from 50.0-100µg/ml and one of the 40.0µg/ml cultures were excessively cytotoxic and were not used in the analysis. The duplicate treatments from 10.0-30.0µg/ml were at most weakly cytotoxic. A small increase in concentration from 30.0-40.0µg/ml resulted in 2.9% survival in one treatment that was available to be cloned. In addition, the relative population growth was significantly reduced at the highest dose analyzed and moderately reduced in one of the 30.0µg/ml treatments (51.7% of control growth). Two of the assayed cultures induced mutant frequencies that were considered elevated but all of the cultures were within the range of acceptable background mutant frequencies. In addition, replicate cultures at the same doses were not significantly elevated and no dose response was observed. A second trial was initiated to confirm the lack of mutagenicity by the test article.

Trial 2 of the nonactivation assay was initiated with nine duplicate treatments from 10.0-60.0µg/ml. Treatments at 50.0µg/ml and 60.0µg/ml were excessively cytotoxic and could not be evaluated for mutant selection and the 10.0µg/ml treatment was terminated because sufficient noncytotoxic doses were available for analysis. The remaining six dose levels induced a wide range of cytotoxicities (109.1% to 24.6% relative survival) and the highest dose had reduced population growth during the expression phase. One of the assayed treatments induced a mutant frequency that was significantly elevated. Again the mutant frequency was within the acceptable range for vehicle controls and the replicate treatment at the same concentration was not significantly elevated. TXIB was therefore evaluated as negative for inducing forward mutations at the HGPRT locus in CHO cells under the nonactivation conditions used in the study.

The mutant frequency of each nonactivation vehicle and positive control was acceptable and within the historical control of the testing laboratory. The assay results achieved all assay acceptable criteria which provided confidence in the assumption that the recorded data represented typical responses of the test article in the nonactivation assay system.

Two mutation assays were initiated with the test article using activation conditions. In trial 1, seven duplicate treatments from 125-2000µg/ml were initiated. Treatments at 125µg/ml were terminated because sufficient noncytotoxic doses were available for analysis. The remaining 6 dose levels were weakly to noncytotoxic. Four treatments were statistically elevated over the vehicle control and none exceeded the acceptable range for the vehicle control cultures. As was observed under nonactivation conditions, duplicates at the same concentration were not significantly elevated and no dose response was observed.

In trial 2, six duplicate treatments from 250-2000µg/ml were initiated and all six dose levels were selected for mutant induction. The analyzed treatments induced little or no cytotoxicity. Trial 2 had two treatments that were significantly elevated and one (1500µg/ml) just exceeded the acceptable range of vehicle control mutant frequency variation. However, a duplicate at the same dose was negative and duplicates at a higher concentration were not elevated. TXIB was therefore evaluated as negative for inducing forward mutations at the HGPRT locus in CHO cells in the presence of S9 metabolic activation.

The mutant frequency of the activation vehicle and positive control were acceptable and within the historical control of the testing laboratory. The assay results achieved all assay acceptable criteria which provided confidence in the assumption that the recorded data represented typical responses of the test article in the assay system.
Remarks on result:
other: all strains/cell types tested
Migrated from field 'Test system'.

Applicant's summary and conclusion

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

2,2,4-Trimethyl-1,3-pentanediol diisobutyrate was not mutagenic in the CHO/HGPRT Forward Mutation Assay when tested at concentrations up to 40 μg/ml in the absence of metabolic activation and 2000 μg/ml in the presence of Aroclor 1254-induced rat liver S9. In a preliminary cytotoxicity assay, the test material was excessively cytotoxic at concentrations of 62.2 μg/ml or greater in the absence of activation but was noncytotoxic at concentrations up to 1990 μg/ml in the presence of activation.
Executive summary:

In a mammalian cell gene mutation assay on the HGPRT locus, CHO cells cultured in vitro were exposed to 2,2,4-trimethyl-1,3-pentanediol diisobutyrate at concentrations of 10, 15, 20, 25, 30, and 40 µg/ml in the absence of metabolic activation and at concentrations of 250, 500, 750, 1000, 1500, 2000 µg/ml in the presence of mammalian metabolic activation (Aroclor 1254 -induced rat liver S9).  In the absence of activation, excessive cytotoxicity was observed at concentrations of 62.2 μg/ml or greater while no cytotoxicity was observed in cultures treated at levels up to 1990 μg/ml in the presence of S9.  The positive controls did induce the appropriate response.  There was no evidence of induced mutant colonies over background for cultures treated with 2,2,4-trimethyl-1,3-pentanediol diisobutyrate.