Bis(2,3-epoxypropyl) terephthalate

Genetic toxicity: in vitro

Administrative data

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

Study period:

August 27, 1992 to October 12, 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Study performed according to OECD guideline 476

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5300 (Detection of Gene Mutations in Somatic Cells in Culture)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Bis(2,3-epoxypropyl) terephthalate

EC Number:

230-565-0

EC Name:

Bis(2,3-epoxypropyl) terephthalate

Cas Number:

7195-44-0

Molecular formula:

C14H14O6

IUPAC Name:

1,4-bis[(oxiran-2-yl)methyl] benzene-1,4-dicarboxylate

Details on test material:

- Name of test material (as cited in study report): TK 12103
- Analytical purity: > 65%
- Lot/batch No.: Z 2204 + Z 2210
- Expiration date of the lot/batch: March 31, 1995

Method

Target gene:

Thymidine kinase gene

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

L5178Y TK+/- mouse lymphoma cells, obtained from the American Type Culture Collection, Rockville, were grown in unstirred suspension culture in growth medium (RM10). The humidity in the incubator was adjusted to >85% rH, the air was enriched to 5 ± 2.0 Vol% C02 and the temperature was 37 ± 1°C. Cultures were maintained in exponential growth by addition of fresh growth medium and were passaged weekly at 5x10E4 cells/ml. Stocks of the mouse lymphoma L5178Y cell line were stored in liquid nitrogen allowing the repeated use of the same cell culture batch in experiments.
Consequently, the parameters of the experiments remain similar because of the reproducible characteristics of the cells. The frozen cell suspension contains 10% dimethylsulfoxide (DMSO). All stock cells have been cultured in cleansing medium for one day to purge the cultures of existing TK-/- mutants. Cleansing medium is growth medium supplemented with 6 µg/ml thymidine, 5 µg/ml hypoxanthine, 7.5 µg/ml glycine and 0.1 µg/ml methotrexate (THMG) . All stock cells were ckecked for mycoplasma contamination, using the Hoechst Dye staining method or the 6-MPDR (6-methylpurine deoxyriboside) method before being frozen. A fresh sample was removed and cultured for each experiment.

Metabolic activation:

with and without

Metabolic activation system:

Rat-liver post mitochondrial supernatant (S9 fraction from male RAI rats (Tif: RAIf[SPF]), treated with Aroclor 1254 (500 mg/kg, i.p.).

Test concentrations with justification for top dose:

Five concentrations were tested in the original and confirmatory experiment:
with S9: 2.54 µg/ml, 5.08 µg/ml, 10.16 µg/ml, 20.32 µg/ml and 40.63 µg/ml
without S9: 0.02 µg/ml, 0.04 µg/ml, 0.08 µg/ml, 0.16 µg/ml and 0.32 µg/ml

Vehicle / solvent:

Acetone

Controls

Controls 1

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

Remarks:

with S9 metabolic activation medium

Controls 2

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without S9 metabolic activation medium

Details on test system and experimental conditions:

Cytotoxicity range-finder:
A range-finding cytotoxicity experiment was performed to establish an appropriate concentration range for the mutation experiments. For each
concentration and the untreated controls, at least 5.0xl0e6 L5178Y cells were seeded in growth medium into each of a series of plastic tissue culture flask and incubated overnight. On the next day the cultures were placed each into sterile disposable 50 ml centrifuge tubes and exposed to the test substance for four hours either in the presence or absence of a metabolic activation system. In the two parts of the experiment, 12 concentrations of the test substance and two vehicle controls were applied. After four hours shaking at 37 °C, the tubes were centrifugea at 200 g for 5-8 minutes, the cells washed with phosphate buffered salineand resuspended in fresh medium (RM10). The cells were counted using a haemocytometer or electronic coulter counter. To assess survival, a sample of each culture was diluted to eight cells/ml immediately post treatment and, for each
concentration, 0.2 ml was plated into two 96-well microtitre plates. The plates were incubated at 37°C in a humidified incubator gassed with 5% v/v
C02 in air for at least 4 days to allow cell growth. Wells containing viable clones were identified under a microscope and counted.

Mutagenicity test:
At least 5.0xl0e6 L5178Y cells were placed in growth medium (RM10) into each of a series of plastic tissue culture flasks and incubated overnight.
The growth medium was replaced for four hours by 36 ml treatment medium (RM5) and 4.0 ml S9 activation mixture, or for 4 hours by 40 ml
treatment medium alone. During this period the cultures were moved on a horizontal shaker at 37°C. In each assay, cultures were treated in duplicate with at least four test chemical concentrations, a positive and a negative control. At the end of the treatment period, the cells were pelleted by
centrifuging at 200 g for 5-8 minutes. The supernatant was discarded and the cells washed with phosphate buffered saline and resuspended in fresh RPMI 1640 medium. The cells were counted using a haemocytometer or electronic coulter counter. Where sufficient cells survived, the concentration was adjusted to 2.5x10e5 cells/ml. To assess survival, a sample of each culture was diluted to eight cells/ml immediately post treatment: 100 µl of the post treated cultures at 2.5x10e5 cells/ml was added to 9.9 ml of medium (RM20) followed by a second dilution of 160 µl (from the 10.0 ml volume) added to 49.84 ml of medium (RM20) . This final 50 ml at eight cells/ml constituted the survival cultures and were dispensed at 200 µl per well into two 96-well microtitre plates (as an average 1.6 cells per well). The plates were incubated (37 °C, 5% C02, >85% rH) for 1-2 weeks to allow cell growth. Wells containg viable clones were identified by eye using background illumination and counted.

The post-treated cultures were returned to the incubator for the expression time of 48 hours. To maintain exponential growth during this phase,
each culture was counted and diluted daily to 2.5x10e5 cell/ml in 40 ml RM10, thereby ensuring 10 cells at each subculture.

At the end of the expression period cultures were plated for viability and 5-trifluorothymidine (TFT) resistance. All cell densities were determined
and the cultures then divided into two series of dilutions as detailed below. A sample of the postexpression culture was diluted to give 100 ml at
1x10e4 cells/ml ('mutation cultures'). From each of this cultures (at 1x10e4 cells/ml) a sample was removed and diluted to eight cells per ml: 500 µl (at 1x10e4 cells/ml) were added to 9.5 ml of medium (RM20) , followed by a second dilution of 800 µl (from the preceding 10.0 ml volume) to 49.2 ml of medium (RM20) . This final 50 ml of eight cells constituted the viability cultures. Each viability culture was dispensed at 200 µl per well into two 96-well microtitre plates (1.6 cells per well). These plates were incubated for 1-2 weeks to allow growth of the cells. Wells containg viable clones were identified by eye using background illumination and counted.

TFT was added to the 'mutation cultures' to give a final concentration of 4 µg/ml. Each TFT treated culture was dispensed at 200 µl per well into four 96-well microtitre plates (2000 cells per well). These plates were incubated for 1-2 weeks to allow cell growth. Wells containg clones were identified as above and counted.

Evaluation criteria:

The test substance was considered to be mutagenic if:
- The assay was valid
- The mutant frequency at one or more concentrations was statistically significant greater than that of the negative control
- There was a statistically significant dose-relationship as indicated by the linear trend analysis
- The effects were reproducible

Statistics:

Statistical significance of mutant frequencies (total wells with clones) was carried out according to the UKEMS guidelines: ROBINSON, W. D., GREEN, M. H. L., COLE, J., HEALY, M. J. R. and GATEHOUSE, D. (1990) Statistical evaluation of bacterial/mammalian fluctuation test.
Thus the control log mutant frequency (LMF) was compared with LMF from each treatment dose, and secondly the data were checked for a linear
trend in mutant frequency with treatment dose. These tests required the calculation of the heterogeneity factor to obtain a modified estimate of
variance.

Results and discussion

Test results

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:

Toxicity
In the cytoxicity range-finder experiment, 12 concentrations of TK 12 103 were tested. In the part with metabolic activation, the concentrations ranged from 0.32 to 650.0 ug/ml separated by 2 fold intervals. Due to complete toxicity in the part without metabolic activation the range-finder had
to be repeated with a different concentration range. The concentrations applied ranged from 0.20 ng/ml to 0.32 ug/ml. Only the data of the
repetition are shown. In the part with metabolic activation at the four highest concentrations no cells survived the treatment with the test chemical. The highest concentration resulting in surviving colonies revealed a relative survival of 34.44%. In the part without metabolic activation the relative survival at the highest concentration was 49.37%. TK 12 103 produced a very pronounced toxic effect with and without metabolic activation.
Accordingly, five concentrations were selected for the original mutagenicity experiment. In the part with metabolic activation the following concentrations were selected: 2.54, 5.08, 10.16, 20.32 and 40.63 ug/ml. At the top concentration of 40.63 ug/ml the mean zero hour survival value was
37.16%. The relative total growth at the end of the expression period revealed a mean value of 16.76% indicating a late toxicity of the test
substance. In the part without metabolic activation the concentrations appplied were: 0.02, 0.04, 0.08, 0.16 and 0.32 ug/ml. The highest
concentration showed a mean relativesurvival of 17.20%. The mean relative total growth value was 6.55%. All concentrations were selected to
determine viability and TFT-resistance 2 days after treatment.
In the confirmatory mutagenicity experiment the identical concentration ranges were selected. The mean relative survival value obtained in the
part with metabolic activation was 12.73% at the highest concentration. The mean relative total growth value obtained after the two days
expression period was 8.62%. Again, this value was lower than the initial toxicity at day zero. The zero hour survival value in the part without
metabolic activation was very low with 1.24%. All concentrations were selected to determine viability and TFT-resistance 2 days after treatment.

Mutation
In the presence of metabolic activation in the original experiment, significant increases in mutant freqency were observed at the three highest
concentrations and a concentration-related effect could be demonstrated. At the lowest concentration of 2.54 ug/ml excessive heterogeneity was
observed between the replicate cultures for the mutation plates and the data were excluded from further analysis. In the confirmatory experiment a
reproducible, statistically significant and concentration-related increase in mutant frequency could be detected for the four highest concentrations.
In the absence of metabolic activation in the original experiment, except for the lowest concentration, all treatment levels showed a statistically
significant increase in mutant frequency when compared to the negative control. The test for linear trend revealed a significant concentration-related effect of the test chemical. The lowest concentration had to be excluded from analysis due to excessive heterogeneity of replicate mutation plates.
In the confirmatory experiment the results could be confirmed. At all concentrations a statistically significant and concentration-related increase in
mutant frequency could be observed.

Colonies sizing:
For the negative controls, the positive controls and for treated cultures with statistically significant increased mutant frequency, the number of wells
containing small colonies and the number of wells containing large colonies were scored.
With metabolic activation the proportion of small colonies in the negative control ranged from 47.5% to 63.4%, whereas with the positive control
(DMN) an increased proportion of 46.2%-51.6% was observed.
Without metabolic activation the proportion of small colonies in the negative control ranged from 29.9% to 51.5%, whereas with the positive control
(EMS) a proportion of 27.0%-52.8% was observed.
In the original and confirmatory experiment with metabolic activation, only the two highest concentrations were scored for large and small colonies.
In both experiments a higher proportion of small colonies was observed. Without metabolic activation the occurrence of small colonies at the highest concentration was very pronounced. At lower concentrations the values found were within the range of the negative control.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive

Based on the results of two independently performed experiments and under the given experimental conditions, it is concluded that
TK 12 103 and its metabolites demonstrated a pronounced ability to induce mutations at the tk locus of mouse lymphoma L5178Y cells in
the presence and absence of metabolic activation.

Executive summary:

TK 12 103 was tested for its ability to induce mutations at the tk locus (5-trifluorothymidine resistance) in L5178Y mouse lymphoma

cells. The study consisted of a preliminary cytotoxicity rangefinder and two independent experiments, each performed with and

without metabolic activation by an Aroclor 1254 induced rat liver post-mitochondrial supernatant (S9 fraction).

In a first range-finder experiment the concentrations applied ranged from 0.32 to 650.00 ug/ml separated by 2-fold intervals.

In the part with metabolic activation the highest concentration resulting in surviving colonies was 40.63 ug/ml yielding a relative survival of 34.44%. In the part without metabolic activation cells did not survive the treatment with TK 12 103 down to the concentration of 0.64 ug/ml. Therefore the part without metabolic activation was repeated with concentrations ranging from 0.20 ng/ml to 0.32 ug/ml. At the top concentration the relative survival was 49.37%.

Accordingly, 5 concentrations were chosen for the first mutagenicity experiment, separated by 2-fold intervals and ranging from 2.54 to 40.63 ug/ml in the part with metabolic activation and from 0.02 to 0.32 ug/ml in the part without metabolic activation. All concentrations were plated for viability and 5-trifluorothymidine resistance two days after treatment. At the highest concentrations the zero hour survival was 37.16% and 17.20% in the presence and absence of the S9 fraction. In the confirmatory experiment the same concentration ranges were

selected. With metabolic activation the relative survival at the highest concentration was 12.73%, without metabolic activation the value found was 1.24%.

Negative (vehicle) and positive control treatments were included in each experiment with and without metabolic activation. The mutant frequencies of the negative controls were within normal ranges and the positive controls N-nitrosodimethylamine (DMN, with S9) and ethylmethansulfonate (EMS, without S9) produced statistically significant increases of mutant frequency.

In the part performed with metabolic activation, reproducible statistically significant and concentration-related increases in mutant frequency were observed in the two experiments over the concentration range of 10.16 to 40.63 ug/ml. In the part without metabolic activation, reproducible statistically significant and concentration-related increases in mutant frequency could be observed over a concentration range of 0.04 to 0.32 ug/ml.

At the highest concentrations tested with and without metabolic activation an increased proportion of small (slow growing) mutant colonies was obtained, indicating the induction of large genetic damage (chromosome aberrations).

Therefore, the results of the Mouse Lymphoma Mutagenicity Assay indicate that, under the conditions of this study,bis(oxiran-2-ylmethyl) terephthalate was concluded to be positive with and without S9 metabolic activation.