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Toxicological information

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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
5 january 2000 - 23 March 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed under GLP and according to internationally accepted guidelines.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
Test article: p-Toluenesulfonamide
Supplier: Akzo Nobel Chemicals Inc.
Date received: January 21, 2000.
Storage: ambient temperature.
Purity: 99.9% (taken from CoA)

Method

Target gene:
The thymidine kinase (TK) locus
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media:
The medium used for this study was RPMI 1640 supplemented with horse serum (10% by volume), Pluronic F68, L-glutamine, sodium pyruvate, penicillin and streptomycin.
Treatment medium was Fischer's medium with the same medium supplements used in the culture medium except that the horse serum concentration was reduced to 5% by volume.
Cloning medium consisted of the RPMI 1640 culture medium with up to 20% horse serum, without Pluronic F68 and with the addition of 0.24% BBL agar to achieve a semisolid state.
Selection medium was cloning medium that contained 3 µg/mL of TFT.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Dose range finding assay with and without S9-mix: 9.85, 19.7, 39.3, 78.5, 157, 313, 625, 1250, 2500, 5000 µg/L
1st Mutation assay with S9-mix: 125, 250, 500, 750, 1000, 1500, 2000, 2500, 3000, 4000 and 5000 µg/mL
2nd Mutation assay with S9-mix: 125, 250, 500, 750, 1000, 1500, 2000, 2250, 2500, 3000 and 4000 µg/mL
1st Mutation assay without S9-mix: 125, 250, 500, 750, 1000, 1500, 2000, 2500, 3000, 4000 and 5000 µg/mL
2nd Mutation assay without S9-mix: 31.3, 62.5, 125, 250, 500, 750, 1000, 1500, 2000, 2500 and 3000 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test article, p-Toluenesulfonamide, was soluble in the vehicle (DMSO) at 500 mg/mL, the highest concentration prepared.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
methylmethanesulfonate
other: see "any other information on materials..."
Details on test system and experimental conditions:
METHOD OF APPLICATION: in suspension

DURATION
- Preincubation period: not applicable
- Exposure duration:
1st mutation assay without S9-mix: 4 hours
2nd mutation assay without S9-mix: 24 hours
1st mutation assay with S9-mix: 4 hours
2nd mutation assay with S9-mix: 4 hours
- Expression time (cells in growth medium):
1st mutation assay without S9-mix: 2 days
2nd mutation assay without S9-mix: 2 days
1st mutation assay with S9-mix: 2 days
2nd mutation assay with S9-mix: 2 days
- Selection time (if incubation with a selection agent):
1st mutation assay without S9-mix: 13 days
2nd mutation assay without S9-mix: 13 days
1st mutation assay with S9-mix: 13 days
2nd mutation assay with S9-mix: 13 days
- Fixation time (start of exposure up to fixation or harvest of cells):
Not applicable

SELECTION AGENT (mutation assays): TFT (5-trifluorothymidin)
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): not applicable


NUMBER OF REPLICATIONS:
The assay conditions consisted of vehicle controls in triplicate, two positive controls and eleven different test article dose levels using one culture per dose level. Treated cultures may be eliminated during the expression period as long as four dose levels are left for analysis of mutant induction. After exposure and expression time cells from each selected tube was suspended in selection medium in soft agar to recover TFT-resistant mutants. Therfore for the selection of mutants the sample was distributed into three 100 mm dishes.

NUMBER OF CELLS EVALUATED: The total number colonies of cells were counted to assess mutant frequency and the number of large and small colonies was recorded since this is considered to reflect the types of genetic damage, with the large colonies derived from cells with intragenic mutations that affect only the TK gene and the small colonies the result of larger mutations that affect cell growth as well as the TK gene.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER EXAMINATIONS:
- Determination of polyploidy: not applicable
- Determination of endoreplication: not applicable
- Other: not applicable

OTHER: not applicable
Evaluation criteria:
Assay Evaluation Criteria
The test article was evaluated as positive, negative, or equivocal in this assay.

Evaluation of a Positive Response.
The test article is evaluated as positive if dose-dependent increases of 2-fold or greater in mutant frequency are obtained over the concurrent background mutant frequency. The background mutant frequency is defined as the average mutant frequency of the vehicle control cultures. The 2-fold or greater increase is based on extensive experience which indicates such responses are repeatable in additional trials. It is desirable to obtain this
relationship for at least three doses, but this goal depends on the dose steps chosen for the assay and toxicity at which mutagenic activity appears.
The dose-dependent requirement is waived if a large increase in mutant frequency (4-fold or higher) is obtained for a single dose at or near the highest testable toxicity. However, for the test article to be evaluated as positive, any increases must be repeatable in a second trial.

Evaluation of a Negative Response.
The test article was evaluated as negative if a 2-fold increase in mutant frequency was not observed for (1) a range of doses that extended to toxicities causing 10% to 20% Relative Total Growth (RTG), or (2) for relatively nontoxic test articles, a range of doses extending to the maximum concentration of 5 mg/mL or 10 mM (whichever is lower), or (3) a range of doses that extended to a level approximately twice the solubility limit in culture medium, or (4) the increase(s) are not repeatable in a confirmatory trial.

Evaluation of an Equivocal Response.
The test article was evaluated as equivocal in this test system if there was no consistent evidence for either a positive or negative evaluation.
Statistics:
Not appicable

Results and discussion

Test resultsopen allclose all
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
-S9 24-hrs: noncytotoxic up to 313 µg/mL, moderate cytotoxicity at 625 µg/mL, high cytotoxicity at 1250 µg/mL, excessive cytotoxicity at 2500 and 5000 µg/mL.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
A positive response was observed at the high dose only.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
+ S9 4-hrs: non- to weakly cytotoxic up to 2500 µg/ml, and excessively cytotoxic at 5000 µg/mL.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH at the top dose of test article prepared in treatment medium (5000 µg/mL) was the same as the pH of the vehicle control. In addition, for all doses there was no color change of the pH indicator in any of the treatments in the medium compared to the vehicle controls.
- Effects of osmolality: Osmolality of the treatment medium was also determined. However, due to the formation of a precipitate, the osmolality of the highest concentration of test article prepared could not be measured. Measurement of the second highest concentration prepared for the cytotoxicity assay (2500 µg/mL) indicated no change when compared to the vehicle control.
- Evaporation from medium: not applicable
- Water solubility: no data
- Precipitation: When the dosing stocks were added to treatment medium plus cells, a precipitate was observed at and above 3000 µg/mL.
- Other confounding effects: no data


RANGE-FINDING/SCREENING STUDIES:
The test article, p-Toluenesulfonamide, was tested in a preliminary dose rangefinding assay with a treatment period of approximately 4 hours both with and without S9 metabolic activation and a preliminary dose rangefinding nonactivation assay with a treatment period of approximately 24 hours. Ten dose levels were used in each case that ranged from 9.85 to 5000 µg/mL; a vehicle control was included under each activation condition.
In the presence and absence of rat liver S9 metabolic activity with a 4-hour treatment period, p-Toluenesulfonamide was noncytotoxic to weakly cytotoxic up to 2500 µg/ml, and excessively cytotoxic at 5000 µg/mL. In the nonactivation dose rangefinding assay using a 24-hour treatment period (Table 2), the test article induced no cytotoxicity up to 3 13 µg/mL, moderate cytotoxicity at 625 µg/mL, high cytotoxicity at 1250 µg/mL, and excessive cytotoxicity at 2500 and 5000 µg/mL. The top dose chosen was 5000 µg/mL, which was the testing limit for the assay.

COMPARISON WITH HISTORICAL CONTROL DATA: Yes

ADDITIONAL INFORMATION ON CYTOTOXICITY: None

Any other information on results incl. tables

Mutation Assays

Initial Nonactivation Mutation Assay.

Eleven concentrations of p-Toluenesulfonamide at 125, 250, 500, 750, 1000, 1500,2000, 2500, 3000,4000 and 5000 µg/mL were initiated. Treatments at and above 2500 µg/mL were terminated due to excessive cytotoxicity. The remaining seven treatments induced weak to high cytotoxicity (76.4% to 19.6% relative growths), In order for a treatment to be evaluated as mutagenic in the initial nonactivation assay, a mutant frequency greater than 94.0 % was required. This threshold value was equal to twice the average mutant frequency of the concurrent vehicle controls (background mutant frequency). None of the analyzed treatment induced this level of mutant action. A confirmatory assay was initiated.

Confirmatory Nonactivation Mutation Assay.

In the confirmatory nonactivation assay, the treatment period was approximately 24 hours. Eleven treatments at 31.3,62.5, 125, 250, 500,750, 1000, 1500,2000,2500 and 3000 µg/mL were initiated. Treatments at and above 1500 µg/mL were terminated due to excessive cytotoxicity.

The remaining seven treatments were selected for mutant analysis and induced weak to high cytotoxicity (70.6% to 11.7% relative growths). None of the analyzed treatments induced a mutant frequency that exceeded the minimum criterion of 122.3 x 10E-6.

The test article is, therefore, considered negative without metabolic activation.

Initial Activation Mutation Assay.

Eleven treatments at 125,250, 500, 750, 1000, 1500,2000, 2500,3000,4000 and 5000 µg/mL were initiated, and treatments at and above 2500 pg/mL were terminated due to excessive cytotoxicity. The remaining seven doses were cloned for mutant analysis and induced no cytotoxicity to moderately high cytotoxicity (133.9% to 29.0% relative growths). The minimum criterion for a positive response in this trial was 99.8 x 10E-6. Treatment at 2000 µg/mL induced this level of mutant action with a mutant frequency that was 2.1-times the average vehicle control mutant frequency. A confirmatory assay was performed.

Confirmatory Activation Mutation Assay.

In the confirmatory assay with metabolic activation, eleven treatments at 125,250,500, 750, 1000, 1500,2000,2250,2500,3000 and

4000 µg/mL were initiated. Treatments at and above 2250 µg/mL were terminated due to excessive cytotoxicity. The remaining seven doses induced no cytotoxicity to high cytotoxicity (116.7% to 14.5% relative growths). Treatment at 2000 µg/mL induced a mutant frequency that

exceeded the minimum criteria of 164.0 x 10E-6 with a mutant frequency that was 3.1 -times the average vehicle control mutant frequency. The test article was evaluated as positive with metabolic activation in this assay.

Control Values.

The average cloning efficiencies for the vehicle controis were 104.4% and 90.8% without activation and 83.8% and 95.8% with S9 metabolic activation, which demonstrated acceptable cloning conditions for the assays. The positive control cultures, MMS (nonactivation) and MCA (activation) induced large increases in mutant frequency that were greatly in excess of the minimum criteria.

Sizing Analysis.

The L5 178Y TK +/- mutation assay produces a bimodal distribution of large and small mutant colonies. The origin of the bimodal distribution of mutant colony sizes is considered to reflect the types of genetic damage, with the large colonies derived from cells with intragenic mutations that affect only the TK gene and the small colonies the result of larger mutations that affect cell growth as well as the TK gene. Colony sizing was performed on all cultures. Mutant colonies from all the cultures showed the expected bimodal distribution and mutant colonies from MMS and MCA treated cultures showed both small and large colonies. An increase in small colonies was observed in treatments which induced a positive response.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive with metabolic activation

The test article, p-Toluenesulfonamide, was evaluated as negative for inducing forward mutations at the TK locus in L5 178Y mouse lymphoma cells under nonactivation conditions. Under activation conditions, a positive response was observed at the highest, cytotoxic, dose only.
Executive summary:

The objective of this in vitro assay was to evaluate the ability of p-Toluenesulfonamide to induce forward mutations at the thymidine kinase (TK) locus in the mouse lymphoma L5 178Y cell line. The study was performed according to OECD476 and under GLP.

The test article, p-Toluenesulfonamide, was soluble in the vehicle (DMSO) at 500 mg/mL, which was the highest concentration prepared. When the dosing stocks were added to treatment medium plus cells, a precipitate was observed at and above 3000 µg/mL.

A dose rangefinding assay was performed with and without metabolic activation using a treatment period of approximately 4 hours. In addition, a dose rangefinding assay without metabolic activation using a treatment period of approximately 24 hours was performed. The dose rangefinding assays were initiated with concentrations from 9.85 to 5000 µg/mL. In the presence and absence of rat liver S9 metabolic activity with a 4-hour treatment period, p-Toluenesulfonamide was noncytotoxic to weakly cytotoxic up to 2500 µg/mL, and excessively cytotoxic at 5000 µg/mL. In the nonactivation dose rangefinding assay using a 24 -hour treatment period, the test article induced no cytotoxicity up to 313 µg/mL, moderate cytotoxicity at 625 µg/mL, high cytotoxicity at 1250 µg/mL, and excessive cytotoxicity at 2500 and 5000 µg/mL.

The top dose chosen for the mutation assays was 5000 µg/mL, which was the testing limit for the assay.

In the nonactivation mutation assay with a treatment period of approximately 4 hours, seven doses ranging from 125 to 2000 µg/mL were analyzed for mutant induction and weak cytotoxicity to high cytotoxicity was induced. None of the analyzed treatments induced a mutant frequency that exceeded the minimum criteria for a positive response.

A confirmatory assay was performed. In the confirmatory nonactivation mutation assay, which was performed with a 24-hour treatment period, seven treatments from 31.3 to 1000 µg/mL were analyzed and weak cytotoxicity to high cytotoxicity was induced. None of the treatments induced a mutant frequency that exceeded the minimum criteria for a positive response.

The test article was, therefore, evaluated as negative without metabolic activation.

(In the first assay at 2000 µg/mL the relative growth was 20% of the control indicating cytotoxicity. The mutation frequency at this dose was almost double of the control. Increase was only in small colonies. This small increase was not observed in the repeat assay)

In the initial mutation assay in the presence of S9 metabolic activation, seven treatments from 125 to 2000 µg/mL were analyzed and no cytotoxicity to moderately high cytotoxicity was induced. In the confirmatory assay, seven treatments from 125 to 2000 µg/mL were also analyzed, and no cytotoxicity to high cytotoxicity was induced. In both the initial and confirmatory assays, treatment at 2000 µg/mL induced a mutant frequency that exceeded the minimum criteria for a positive response with mutant frequencies 2.1- and 3.1 -times the average vehicle control values, respectively.

The test article was evaluated as positive with metabolic activation.

In both assyas the incraese was only obsreved at the highest (cytotoxic) dose and involved an incrase in small colonies only.

The test article, p-Toluenesulfonamide, was evaiuated as negative for inducing forward mutations at the TK locus in L5178Y mouse lymphoma cells under nonactivation conditions. Under activation conditions, a positive response was observed at the highest, cytotoxic, dose only.