1-[2-(2-chloroethoxy)phenylsulfonyl]-3-(4-methoxy-6-methyl-1,3,5-triazin-2-yl)urea;triasulfuron (ISO)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 Mar 2010 to 28 Jun 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1997

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

1998

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

2008

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Test material

Test material information

Constituent 1

Reference substance name:

2-(2-chloroethoxy)-N-[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)carbamoyl]benzene-1-sulfonamide

EC Number:

617-298-9

Cas Number:

82097-50-5

Molecular formula:

C14H16ClN5O5S

IUPAC Name:

2-(2-chloroethoxy)-N-[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)carbamoyl]benzene-1-sulfonamide

Method

Target gene:

Thymidine Kinase

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Stock cultures are propagated in plastic flasks in RPMI 1640 complete culture medium. The cells are subcultured two times prior to treatment. The cell cultures are incubated at 37 ± 1.5°C in a humidified atmosphere with 4.5 % carbon dioxide and 95.5 % ambient air.

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/B-naphthoflavone induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from 8 - 12 week old male Wistar HsdCpb:WU rats weight approx. 220 - 320 g induced by applications of 80 mg/kg bw phenobarbital and B-naphthoflavone each on three consecutive days. The livers were prepared 24 hours after the last treatment. The S9 fractions were produced by dilution of the liver homogenate with a 150 mM KCl solution (1/4, v/v) followed by centrifugation at 9000 × g. Aliquots of the supernatant were frozen and stored in ampoules at approx. -80° C. Small numbers of the ampoules were kept at -20°C for up to one week. Each batch of S9 mix was routinely tested with 2-aminoanthracene as well as benzo[a]pyrene. The protein concentration of the S9 preparation was 34.4 mg/mL in the pre-experiment and in experiment I, and 35.0 mg/mL in experiment II. An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to give a final protein concentration of 0.75 mg/mL in the cultures. The concentration in the final test medium was 5 % (v/v).

Test concentrations with justification for top dose:

Experiment I and II:
With & without S9 mix: 420; 840; 1680; 3360; 4280 μg/mL

Vehicle / solvent:

DMSO 1 % (v/v).

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

DOSING PREPARATIONS
On the day of the experiment (immediately before treatment), the test item was dissolved or suspended in DMSO. The final concentration of DMSO in the culture medium was 1 % (v/v).

EXPERIMENTAL DESIGN
The assay was performed in two independent experiments, using two parallel cultures each. The main experiments were performed with and without liver microsomal activation and a treatment period of 4 hours. The concentration range of the main experiments went up to approximately 10 mM. Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid.

CELL PREPARATION
Prior to mutagenicity testing the amount of spontaneous mutants was reduced by growing the cells for one day in RPMI 1640-HAT medium supplemented with: hypoxanthine 1.0×10^-4 M, aminopterin 2.0×10^-7 M, thymidine 1.6×10^-5 M. The incubation of the cells in HAT-medium was followed by a recovery period of 2 days in RPMI 1640 medium containing: hypoxanthine 1.0×10^-4 M, thymidine 1.6×10^-5 M. After this incubation the L5178Y cells were returned to normal RPMI 1640 medium (complete culture medium). Large stocks of the cleansed L5178Y cell line are stored in liquid nitrogen in the cell bank allowing the repeated use of the same cell culture batch in many experiments. Before freezing, each batch was screened for mycoplasma contamination and checked for karyotype stability. Consequently, the parameters of the experiments remain similar because of the reproducible characteristics of the cells. Thawed stock cultures are propagated in plastic flasks in RPMI 1640 complete culture medium. The cells are subcultured two times prior to treatment. The cell cultures are incubated at 37 ± 1.5°C in a humidified atmosphere with 4.5 % carbon dioxide and 95.5 % ambient air.

CULTURE TREATMENT
In the mutation experiment 1×10^7 cells/flask (80 cm2 flasks) suspended in 10 mL RPMI medium with 3 % horse serum were exposed to various concentrations of the test item either in the presence or absence of metabolic activation. After 4 h the test item was removed by centrifugation and the cells were washed twice with "saline G". Subsequently the cells were resuspended in 30 mL complete culture medium and incubated for an expression and growth period of 48 h. The cell density was determined each day and adjusted to 3×10^5 cells/mL, if necessary. The relative suspension growth (RSG) of the treated cell cultures was calculated by the day 1 fold-increase in cell number multiplied by the day 2 fold-increase in cell number according to the method of Clive and Spector. One sample of the cells was taken at the end of treatment, diluted and seeded into microtiter plates, to determine the viability of the cells after treatment (cloning efficiency 1). After the expression period the cultures were selected. Cells from each experimental group were seeded into 2 microtiter plates so that each well contained approximately 4×10^3 cells in selective medium with TFT. The viability (cloning efficiency 2) was determined by seeding about 2 cells per well into microtiter plates (same medium without TFT). The plates were incubated at 37 ± 1.5 °C in 4.5 % CO2/95.5 % water saturated air for 10 - 15 days. Then the plates were evaluated.

SIZE DISTRIBUTION OF THE COLONIES:
Colonies were counted manually. In accordance with their size the colonies were classified into two groups. The colony size distribution was determined in the controls and at all concentrations of the test item. Criteria to determine colony size were the absolute size of the colony (more than 1/3 of a well for large colonies) and the optical density of the colonies (the optical density of the small colonies is generally higher than the large colonies).

SURVIVAL
The survival rate and viability were determined based on the Poisson distribution method. The zero term of the Poisson distribution, [P(0)] method, was used. The mutation frequency was derived from the cloning efficiency under selective conditions compared to the corresponding viability under non-selective conditions.

Evaluation criteria:

A test item is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 10^6 cells above the corresponding solvent control. A relevant increase of the mutation frequency should be dose-dependent. A mutagenic response is considered to be reproducible if it occurs in both parallel cultures. However, in the evaluation of the test results the historical variability of the mutation rates in negative and vehicle controls and the mutation rates of all negative and vehicle controls of this study are taken into consideration. Results of test groups are generally rejected if the relative total growth, and the cloning efficiency 1 is less than 10 % of the vehicle control unless the exception criteria specified by the IWGT recommendations are fulfilled. Whenever a test item is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used to differentiate point mutations from clastogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and dose dependent shift in the ratio of small versus large colonies clastogenic effects are indicated. A test item is classified as non-mutagenic if the induced mutation frequency does not reproducibly exceed a threshold of 126 colonies per 10^6 cells above the corresponding solvent control or negative control, respectively. A test item not meeting the conditions for a classification as mutagenic or non-mutagenic will be considered equivocal in this assay and may be considered for further investigation.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT11 statistics software. The number of mutant colonies obtained for the groups treated with the test item was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological relevance and statistical significance were considered together.

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Isolated in one of the parallel cultures was noted in the first experiment at 4280 μg/mL with metabolic activation

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

CYTOTOXICITY:
No relevant cytotoxic effects indicated by a relative cloning efficiency 1 (survival) or a relative total growth (RTG) of less than 50% in both cultures occurred. An isolated cytotoxic effect in one of the parallel cultures was noted in the first experiment at 4280 μg/mL with metabolic activation. Precipitation of the test item was noted at 3360 and 4280 μg/mL in both experiments without metabolic activation. In the experimental parts with metabolic activation precipitation occurred at 1680 Sg/mL and above in the first, and at 3360 and 4280 μg/mL in the second experiment.

MUTAGENICITY:
No substantial and reproducible dose dependent increase of the mutation frequency exceeding the threshold of 126 above the corresponding solvent control was observed in the main experiments with and without metabolic activation. A single, isolated increase exceeding the threshold occurred in the second culture of the second experiment without metabolic activation at a precipitating concentration of 3360 μg/mL. However, this increase was not reproduced in the parallel culture performed under identical experimental conditions. Furthermore, the increase of the mutation frequency above the threshold was not dose dependent as indicated by the lacking statistical significance and was consequently judged as biologically irrelevant artefact caused by precipitation.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT statistics software. No significant trend was observed in any of the experimental parts with and without metabolic activation.

In this study the range of the solvent controls was from 100 up to 196 mutant colonies per 10^6 cells; the range of the test groups treated with the test item was from 69 up to 322 mutant colonies per 10^6 cells. The highest solvent controls (196 and 188 colonies per 10^6 cells) exceeded the 50 – 170 x 10^6 control range as stated under paragraph 3.12, acceptability of the assay of this report but are within the limits recommended in reference 11. Additionally, the mutant frequency of the parallel cultures was fully acceptable (100 and 145 mutant colonies/10^6 cells). The cloning efficiency in culture II of the first experiment with metabolic activation fell just short of the lower limit of 65%. In the first culture of the second experiment without metabolic activation the cloning efficiency exceeded the upper limit of 120%. Both sets of data are acceptable however, since the cloning efficiency of the parallel cultures remained within the acceptable range.

MMS (19.5 μg/mL) and CPA (3.0 and 4.5 μg/mL) were used as positive controls and showed a distinct increase in induced total mutant colonies at acceptable levels of toxicity with at least one of the concentrations of the controls. In the second culture of the second experiment the positive controls with metabolic activation did not quite meet the acceptance criterion of at least 150 induced small colonies. The positive controls in the presence of metabolic activation however, were judged as valid since the positive control at 4.5 μg CPA per mL in culture II showed a substantial increase of total colonies and the corresponding
positive control of culture I easily met the acceptance criteria.

Applicant's summary and conclusion

Conclusions:

In conclusion it can be stated that during the mutagenicitiy test described and under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.

Executive summary:

An OECD 476 study was performed under GLP to investigate the potential of the test item to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. The assay was performed in two independent experiments, using two parallel cultures each. Experiment I and II were performed with and without liver microsomal activation and a treatment period of 4 hours. Both main experiments were performed with and without liver microsomal activation and a treatment period of 4 hours. The concentrations of the test material in the Experiment I and II, with and without S9 mix were 420; 840; 1680; 3360; 4280 μg/mL.
No relevant cytotoxic effects indicated by a relative cloning efficiency 1 (survival) or a relative total growth (RTG) of less than 50% in both cultures occurred in both main
experiments with and without metabolic activation. No substantial and reproducible dose dependent increase in mutant colony numbers was observed with and without metabolic activation. Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid.
In conclusion it can be stated that during the mutagenicity test described and under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation. Therefore, the test item is considered to be non mutagenic in this mouse lymphoma assay.