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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:
experimental study
Adequacy of study:
key study
Study period:
August 12, 1992 to December 14, 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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
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

Constituent 1
Chemical structure
Reference substance name:
Tris(oxiranylmethyl) benzene-1,2,4-tricarboxylate
EC Number:
230-638-7
EC Name:
Tris(oxiranylmethyl) benzene-1,2,4-tricarboxylate
Cas Number:
7237-83-4
Molecular formula:
C18H18O9
IUPAC Name:
tris(oxiranylmethyl) benzene-1,2,4-tricarboxylate
Constituent 2
Reference substance name:
tris(oxiranylmethyl)benzene-1,2,3-tricarboxyl
IUPAC Name:
tris(oxiranylmethyl)benzene-1,2,3-tricarboxyl
Details on test material:
- Name of test material (as cited in study report): TK 30009 (LMB 5196)
- Analytical purity: > 75%
- batch No.: Z 2178
- Expiration date of the lot/batch: March 1995
- Stability: Stable

Method

Target gene:
Thymidine kinase gene (5-trifluorothymidine resistance)
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.

Additional strain / cell type characteristics:
not specified
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:
In the original experiment with S9: 2.25 µg/ml, 4.50 µg/ml, 9.00 µg/ml, 18.00 µg/ml and 36.00 µg/ml
In the original experiment without S9: 0.02 µg/ml, 0.04 µg/ml, 0.08 µg/ml, 0.15 µg/ml and 0.30 µg/ml
In the confirmatory experiment with S9: 2.25 µg/ml, 4.50 µg/ml, 9.00 µg/ml, 18.00 µg/ml and 54.00 µg/ml
In the confirmatory experiment without S9: 0.02 µg/ml, 0.04 µg/ml, 0.08 µg/ml, 0.15 µg/ml and 0.45 µg/ml
Vehicle / solvent:
Acteone
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without S9 metabolic activation medium
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-dimethylnitrosamine
Remarks:
with 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
Positive controls validity:
valid
Additional information on results:
Toxicity:
In the cytoxicity range-finder experiment, 12 concentrations of TKK 30009 were tested. In the part with metabolic activation, the concentrations
ranged from 0.57 to 1170.00 µg/ml separated by 2 fold intervals. Due to a marked toxicity down to a concentration of 1.14 µg/ml in the part without
metabolic activation the rangefinder had to be repeated with a different concentration range.
The concentrations applied ranged from 0.30 µg ml to 0.60 µg/ml. 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 3.16%. In the
part without metabolic activation the relative survival at the highest concentration was 8.97%. TKK 30009 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: 2.25, 4.50, 9.00, 18.00 and 3 6.00 µg/ml. At the top concentration of 36.00 µg/ml the mean zero hour survival value
was 46.73%. The relative total growth at the end of the expression period revealed a mean value of 46.29%. In the part without metabolic activation
the concentrations appplied were: 0.02, 0.04, 0.08, 0.15 and 0.30 µg/ml. The highest concentration showed a mean relative survival of 52.06%.
The mean relative total growth value was 53.88%. All concentrations were selected to determine viability and TFT-resistance 2 days after treatment.

Due to a not very pronounced toxicity at the highest concentration the top concentration was enlarged by a factor of 1.5 in the confirmatory
mutagenicity experiment. With metabolic activation the concentrations applied were: 2.25, 4.50, 9.00, 18.00 and 54.00 µg/ml. In the part without
activation the concentrations selected were 0.02, 0.04, 0.08, 0.15 and 0.45 µg/ml . The mean relative survival value obtained in the part with
metabolic activation was 3.58% at the highest concentration. The mean relative total growth value obtained after the two days expression period was 7.49%. The zero hour survival value in the part without metabolic activation was 14.87% and the relative survival 4.90%. 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. In the confirmatory experiment a reproducible, statistically significant andconcentration-related increase in mutant frequency could be detected for the three highest concentrations. In the absence of metabolic activation in the original experiment, the two highest concentrations of 0.30 µg/ml and 0.15 µg/ml 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. In the
confirmatory experiment the results could be confirmed. At the four highest concentrations a statistically significant and concentration-related
increase in mutant frequency could be observed. The increase in mutant frequency was more pronounced than in the original experiment and was
seen over a wider concentration range.

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 22.1% to 25.3%, whereas with the positive control
(DMN) an increased proportion of 40.0%-47.7% was observed.
Without metabolic activation the proportion of small colonies in the negative control ranged from 24.3% to 34.0%, whereas with the positive control
(EMS) a proportion of 21.7%-26.4% was observed.
In the original experiment, with and without metabolic activation the proportion of small colonies of test chemical treated cultures differed not very much from the negative control. In the confirmatory experiment, with and without metabolic activation a higher proportion of small colonies at the
highest concentration was observed. Without metabolic activation the occurrence of small colonies was more 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 TKK 30 009 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:

TKK 30009 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 range-finder 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 0.57 to 1170.00 µg/ml separated by 2-fold intervals.

In the part with metabolic activation the highest concentration resulting in surviving colonies was 73.13 µg/ml yielding a relative survival of 3.16%. In the part without metabolic activation cells did not survive the treatment with TKK 30009 down to the concentration of 1.14 µg/ml. Therefore the part without metabolic activation was repeated with concentrations ranging from 0.30 ng/ml to 0.60 µg/ml. At the top concentration the relative survival was 8.97%.

Accordingly, 5 concentrations were chosen for the first mutagenicity experiment, separated by 2-fold intervals and ranging from

2.25 to 36.00 µg/ml in the part with metabolic activation and from 0.02 to 0.30 µg/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 46.73% and 52.06% in the presence and absence of the S9 fraction. Due to a not very pronounced zero hour

toxicity in the original experiment, in the confirmatory experiment the highest concentrations were chosen to be 54.00 µg/ml and

0.45 µg/ml with and without metabolic activation. The four lower concentrations were the same as in the original experiment. With

metabolic activation the relative survival at the highest concentration was 3.58%, without metabolic activation the value found was 14.87%.

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 at concentrations of 9.00 µg/ml and higher. In the original experiment,

without metabolic activation, statistically significant and concentration- related increases in mutant frequency could be observed

at concentrations of 0.15 µg/ml and 0.3 0 µg/ml. In the confirmatory experiment statistically significant and concentration-related

effects could be observed down to a concentration of 0.04 µg/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, tris(oxiranylmethyl)benzene-1,2,3-tricarboxyl was concluded to be positive with and without S9 metabolic activation.