2-[-N-(2-cyanoethyl)-4-[(p-nitrophenyl)azo]anilino]ethyl benzoate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 October 1998 to 23 October 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from rat liver

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 50, 160, 500, 1600, 5000 µg/plate
Concentration range in the main test (without metabolic activation): 50, 160, 500, 1600, 5000 µg/plate

Vehicle / solvent:

Solvent: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without metabolic activation: sodium azide (TA 100, TA 1535), 9-aminoacridine hydrochloride hydrate (TA 1537), 2-nitrofluorene (TA98), 1-methyl-3-nitro-1-nitrosoguanidine (WP2uvrA) With metabolic activation: 2-aminoanthracene – all strains.

Details on test system and experimental conditions:

Preparation and storage of a liver homogenate fraction (S9)

The S9 fraction was prepared by the department conducting the study according to Ames et. al (1975). Male Sprague Dawley rats (200-300 g), supplied by Harlan Winkelmann, Gartenstrasse 27, 33178 Borchen, Germany, received a single intraperitoneal injection of Aroclor 1254 (500 mg/kg body weight) 5 days before killing. The livers were removed from at least 5-6 animals at approx. 0 to 4 °C using cold sterile solutions and glassware, and were then pooled and washed in approx. 150 mM KCI (approximately 1 ml/g wet liver). The washed livers were cut into small pieces and homogenized in three volumes of KCI. The homogenate was centrifuged at approx. 9000 g for 10 minutes. The supernatant was the S9 fraction. This was divided into small portions, rapidly frozen and stored at approx. - 80 °C. The protein content was determined for every batch. Also for every batch of S9 an independent validation was performed with a minimum of two different mutagens, e.g., 2-aminoanthracene and dimethylbenzanthracene to confirm metabolic activation by microsomal enzymes.

Preparation of S9-mix

Sufficient S9 fraction was thawed immediately at room temperature before each test. One volume of S9 fraction (batch no. 98/5 for the experiment, protein concentration 24.8 g/l) was mixed with 9 volumes of the S9 cofactor solution, which was kept on ice until used. This preparation is termed S9-mix. The concentrations of the different compounds in the S9-mix were:
8 mM MgCI2 33 mM KCI 5 mM glucose-6-phosphate 4 mM NADP 100 mM phosphate buffer pH 7.4

Bacteria

The strains of Salmonella typhimurium were obtained from Professor B.N. Ames, University of California, U.S.A.. The strain of £ coli was obtained from the National Collection of Industrial Bacteria, Aberdeen, Scotland.
Bacteria were grown overnight in nutrient broth (25 g Oxoid Nutrient Broth No. 2 /liter) at approx. 37 °C. The amount of bacteria in the cell suspension was checked by nephelometry. Inoculation was performed with stock cultures which had been stored at approx. -80 °C. The different bacterial strains are checked half-yearly with regard to their respective biotin, histidine and/or tryptophan requirements, membrane permeability, ampicillin resistance, crystal violet sensitivity, UV resistance and response to diagnostic mutagens. All criteria for a valid assay were fulfilled as described (2, 3).

Assay procedure

The mutation test was performed in both the presence and absence of S9-mix using all bacterial tester strains and a range of concentrations of the test substance. Positive and negative controls as well as solvent controls were included in each test. Triplicate plates were used.
The mutation experiment also assessed the toxicity of the test substance by evaluation of the bacterial lawn in order to select a suitable range of dose levels for a second mutation test. The highest concentration was usually 50 mg/ml of the test substance in the chosen solvent, which provided a final concentration of 5000 ug/plate. Further dilutions of 1600, 500, 160 and 50 pg/plate were used.
A reduced rate of spontaneously occurring colonies and visible thinning of the bacterial lawn were used as toxicity indicators. Thinning of the bacterial lawn was evaluated microscopically.
If the total number of concentrations selected for evaluation in the plate incorporation test does not allow for a statement of genotoxicity of the test substance to be made, an additional plate incorporation test, based on the toxicity results of the first test, has to be performed. If negative or equivocal results obtained, a second mutation experiment was performed on the basis of toxicity results in the plate incorporation test as a preincubation test.

For mutagenicity testing top agar was prepared for the Salmonella strains by mixing 100 ml agar (0.6 % (w/v) agar, 0.5 % (w/v) NaCI) with 10 ml of a 0.5 mM histidine-biotin solution. With E. coli histidine was replaced by tryptophan (2.5 ml, 0.5 mM). The following ingredients were added (in the following order) to 2 ml of molten top agar at approx. 48 °C:

0.5 ml S9-mix (if required) or buffer
0.1 ml of an overnight nutrient broth culture of the bacterial tester strain
0.1 ml test compound suspension (suspended in DMSO)

After mixing, the liquid was poured into a petri dish containing a 25 ml layer of minimal agar (1.5 % (w/v) agar, Vogel-Bonner E medium with 2 % (w/v) glucose). After incubation for approximately 48 hours at approx. 37 deg C in the dark, colonies (his* and trp* revertants) were counted with an automatic colony counter (Artec counter Model 880). The counter was calibrated for each test by comparison of manual count data of three control plates with automatic data of the colony counter. A correction factor was determined to compensate for differences between manual and automatic count. This correction factor was used to automatically adjust the observed number of colonies on each plate to more accurately reflect the actual number of colonies present.

Evaluation criteria:

Criteria for a valid assay
The assay is considered valid if the following criteria are met:
- the solvent control data are within the laboratory's normal control range for the spontaneous mutant frequency
- the positive controls induced increases in the mutation frequency which were both statistically significant and within the laboratory's normal range

Criteria for a positive response
A test compound is classified as mutagenic if it has either of the following effects:
a) it produces at least a 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn
b) it induces a dose-related increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control in at least two to three concentrations of the test compound at complete bacterial background lawn.

If the test substance does not achieve either of the above criteria, it is considered to show no evidence of mutagenic activity in this system.

Statistics:

As above.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Highest concentration 1600 µg/plate

Vehicle controls validity:

other: Historical data

Untreated negative controls validity:

not valid

Positive controls validity:

other: Historical data

Additional information on results:

Sterility checks and control plates

Sterility of S9-mix and the test compound were indicated by the absence of contamination on the test materia! and S9-mix sterility check plates. Control plates (background control and positive controls) gave the expected number of colonies, i.e. values were within the laboratory's historical control.

Solubility and toxicity

The test compound was suspended in DMSO and a stock solution of 50 mg/ml was prepared for the highest concentration, which provided a final concentration of 5000 ug/plate. Further dilutions of 1600, 500, 160 and 50 ug/plate were used in the mutation experiment as the plate incorporation method.

Visible precipitation of the test compound on the plates was observed at 500 ug/plate and above.

Because of heavy precipitation of the test compound the bacterial lawn could only be evaluated at the dose level of 1600 ug/plate and lower doses.
The test compound proved to be not toxic to the bacterial strains in the mutagenicity experiment.

Mutagenicity

In the mutation test T-9601 was tested for mutagenicity with the same concentrations as described in section 5.3. The number of colonies per plate with each strain as well as mean values of 3 plates are given.
In the absence of the metabolic activation system the test compound induced a significant and dose-dependent increase in the number of revertant colonies with the bacterial strains TA 98, TA 100, TA 1537. In the presence of metabolic activation the test compound resulted in relevant increases in the number of revertant colonies with the Salmonella strains TA 98, TA 100, TA 1535, TA 1537. No increases of the revertants were induced at the Eschericia coli strain WP 2uvrA with and without S9-mix.

All positive controls produced significant increases in the number of revertant colonies. Thus, the sensitivity of the assay and the efficacy of the exogenous metabolic activation system were demonstrated.

CONCLUSION

The results lead to the conclusion that T-9601 is mutagenic in these bacterial test systems with and without an exogenous metabolizing system.

Remarks on result:

other: other: preliminary test

Remarks:

Migrated from field 'Test system'.

Conclusions:

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

The results lead to the conclusion that the substance is mutagenic in these bacterial test systems with and without an exogenous metabolizing system

Executive summary:

Study data conducted to EEC-Guideline B.13 & B14 of the Directive 92/68/EEC, OECD Guidleines for Testing of Chemicals 471 and US.EPA; OPPTS 870.5100 Heath Effects Test Guidelines in compliance with GLP.

The results lead to the conclusion that the susbstance is mutagenic in the bacterial systems with and without an exogenous metoblizing system. However it should be noted that the specific method according to Privall - Mitchell for azo dyes was not utilised in this study; hence the results may be due to redundancies within the method employed. This statement is further enforced by the negative mutagenicity results observed in the other in vitro studies conducted.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 November 1998 to 15 January 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

chromo¬somal aberrations in cultured mammalian cells

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Culture medium: MEM (minimal essential medium) with Hanks-salts and 25 mM Hepes-buffer

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix

Test concentrations with justification for top dose:

The test compound was suspended in DMSO and tested at the following concentrations:

First experiment with 3 h treatment time:
without S9-mix: 25, 50, 75, 125*, 250, 500*, 750 and 1285* μg/ml
with S9-mix: 25, 50, 75, 125*, 250, 500*, 750 and 1285* μg/ml

Second experiment with 20 h treatment time:
without S9-mix: 5, 10, 25*, 50, 75*. 125 and 250* μg/ml

Third experiment with 3 h treatment time:
with S9-mix: 1285* μg/ml

*= evaluated slides

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other:

Remarks:

without metabolic activation: EMS (ethyl methane sulfonate), with metabolic activation: CPA (cyclophosphamide) = Endoxan®

Details on test system and experimental conditions:

Reference Compounds
Without metabolic activation
Name or number of compound (I.N.N. orU.S.A.N): ethyl methane sulfonate
Synonyms: EMS
Formula of the compound: C3HB03S
CAS-Register number: 62-50-0
Product number / Code: 820774
Supplier of reference compound: Dr. Theodor Schuchardt & Co., Germany
Batch number: 40606721
Certificate of analysis: certificated by the supplier, Analytical Department, Dr. Bolkart dated December 18th, 1995

With metabolic activation
Name or number of compound {I.N.N. orU.S.A.N): cyclophosphamide
Synonyms: Endoxan®
Formula of the compound: C7H15CI2N2P.H20
CAS-Register number: 50-18-0
Supplier of reference compound: ASTA Medica AG, Germany
Batch number: 603575 B
Certificate of analysis: certificated by the supplier, Quality Control, Dr. Weiss dated March 25th, 1998

Test groups
Treatment time 3h
Without S9-mix with S9-mix
Solvent control: 0.0μg/ml 0.0μg/ml
Positive control: EMS 1500.00μg/ml CPA 3.0μg/ml
Test group 1: 25μg/ml # 20μg/ml #
Test group 2: 50μg/ml # 50μg/ml #
Test group 3: 75μg/ml # 75μg/ml #
Test group 4: 125μg/ml * 125μg/ml *
Test group 5: 250μg/ml # 250μg/ml #
Test group 6: 500μg/ml * 500μg/ml *
Test group 7: 750μg/ml # 750μg/ml #
Test group 8: 1285μg/ml * 1285μg/ml *

Treatment time 20h
Without S9-mix
Solvent control: 0.0μg/ml
Positive control: EMS 400.0μg/ml
Test group 1: 5μg/ml #
Test group 2: 10μg/ml #
Test group 3: 25μg/ml *
Test group 4: 50μg/ml #
Test group 5: 75μg/ml *
Test group 6: 125μg/ml #
Test group 7: 250μg/ml *

*=evaluated slides
#= not used because higher concentrations were evaluated

Control groups:
Solvent controls: cultures treated with the solvent
Positive controls: without metabolic activation: EMS (ethyl methane sulfonate)
with metabolic activation: CPA (cyclophosphamide) = Endoxan®

Formulation of test compound: suspended in DMSO at appropriate concentrations immediately before use

Formulation of reference compounds
EMS dissolved in cell culture medium on the day of treatment, final concentration: 1.5 mg/ml (3 h treatment) final concentration: 0.4 mg/ml (20 h treatment)
CPA dissolved in cell culture medium on the day of treatment, final concentration in ceil culture medium: 3.0 μg/ml

Source of biological material: cell bank of "Genetic Toxicology", HMR Germany, ProTox

Test organism: cell line V79 of Chinese hamster lung fibroblasts

Cell culture medium: MEM (minimal essential medium) with Hanks-salts and 25 mM Hepes-buffer

Experimental conditions in vitro: approx. 37 °C and approx. 4 % C02 in plastic flasks

Preparation and storage of a liver homogenate fraction (S9)
The S9 fraction was prepared by the testing facility according to Ames et. al (1975). Male Sprague Dawley rats (200-300 g), supplied by Harlan Winkelmann, Gartenstrasse 27, 33178 Borchen, Germany, received a single intraperitoneal injection of Aroclor 1254 (500 mg/kg body weight) 5 days before killing. The livers were removed from at least 5-6 animals at approx. 0 to 4 °C using cold sterile solutions and glassware, and were then pooled and washed in approx. 150 mM KCl (approximately 1 ml/g wet liver). The washed livers were cut into small pieces and homogenized in three volumes of KCl. The homogenate was centrifuged at approx. 9000g for 10 minutes. The supernatant, the S9 fraction, was divided into small portions, rapidly frozen and stored at approx. - 80 °C for not longer than six months. The protein content was determined for every batch. Also for every batch of S9 an independent validation was performed with a minimum of two different mutagens, e.g., 2-aminoanthracene and dimethylbenzanthracene to confirm metabolic activation by microsomal enzymes.

Preparation of S9-mix
Sufficient S9 fraction was thawed to room temperature immediately before each test. An appropriate quantity of S9 fraction (batch no. 98/1, protein concentration 51.8 g/l) was mixed with S9 cofactor solution to yield a final protein concentration of 0.3 mg/ml in the cultures which was kept on ice until used. This preparation is termed S9-mix. The concentrations of the different cofactors of the S9-mix were:

8 mM MgCl2
33 mM KCI
5 mM glucose-6-phosphate
5 mM NADP
100 mM phosphate buffer pH 7.4

Cell culture
Large stocks of the mycoplasma-free V79 cell line are stored in liquid nitrogen in the cell bank of "Genetic Toxicology", thus permitting repeated use of the same ceil culture batch for numerous experiments. The identical characteristics of the cells ensure comparability of the experimental parameters.
Thawed stock cultures were kept at approx. 37 °C and approx. 4 % C02 in 175 cm2 plastic flasks. About 5 x 10 5 to 1 x 10 6 cells were seeded into each flask in 30 ml of MEM-medium supplement with approx. 10 % (v/v) FCS (fetal calf serum) containing approx. 2 mM L-glutamine and approx. 0.1 % (w/v) neomycinsulfate. The cells were subcultured twice a week.

Toxicity experiments and dose range finding
A preliminary toxicity test was undertaken in order to select appropriate dose levels for the cytogenetic assay. Cell cultures were subjected to the same treatment conditions as in the main experiment. Cytotoxic effects were determined by photometric measurement of V79 cell cultures grown in microwell plates and stained with crystal violet. The relative cell density in the microwell plates was nearly the same as in the Quadriperm® dishes.

The test included the following treatments:

Solvent control : the maximum final concentration of organic solvents was approx. 1 % (v/v).
Test compound : the highest dose level for the preliminary toxicity test was determined by the solubility of the test compound up to the maximum of 10 mM or 5000μg/ml.
Treatments were performed both in the presence and absence of S9 metabolic activation system using a duplicate cell culture at each test point.

Rationale for dose selection
The concentrations for the mutagenicity assay were based on the results of the toxicity experiment.
For non-toxic, freely soluble test compounds, the top dose is 10 mM or 5000μg/ml according to international testing guidelines.
For relatively insoluble test compounds, that are not toxic at concentrations lower than the insoluble concentration, the highest dose used should be a concentration above the limit of solubility in the final culture medium after the end of the treatment period. In the case of toxic effects, the highest dose level should reduce the survival rate to approximately 20 - 50 % and/or the mitotic index to approximately 50 % compared with the corresponding solvent control.
For toxic compounds additional concentrations may be included in the treatment series. According to the criteria described above, three adequately spaced dose levels extending over at least one decadic logarithm were evaluated. In the event of clearly positive results at the 3 hours treatment time, it is not necessary to perform an evaluation of the 20 h treatment time.
For each experimental point two cultures were used for each concentration.

Mutagenicity test
Two independent experiments were conducted. The first experiment with 3 hours treatment time of the test substance was performed in the presence and the absence of S9-mix. Cultured cells were seeded on-to slides (duplicate culture) then treated for either 3 hours (with and without S9-mix in the first experiment) or for 20 hours (without S9-mix in the second experiment). Colcemide was then added to arrest cell division and the chromosomes were stained and examined. In both assays, cells were sampled 20 hours after the start of treatment. For both assays, at least three dose levels were evaluated for chromosome aberrations. Where negative or equivocal results were obtained, cells were treated and also examined 20 hours after the start of treatment.

Before treatment, the pH values and osmolality of the treatment medium were determined. If necessary the pH was adjusted to pH 7.3 with NaOH or HCl. Any effects on the osmolality during the study were described in the study report.

Two-day old, exponentially growing stock cultures which were over 50 % confluent were trypsinised and a single cell suspension (culture) was prepared. The trypsin concentration was approx. 0.25 % (v/v) in Ca-Mg-free salt solution. Two slides were placed in Quadripem dishes which were then seeded with cells to yield 2-3 x 10 4 cells/slide. Thus for each dose level and treatment time, duplicate cultures were used. The Quadriperm® dishes contained 5 ml MEM with approx. 10 % (v/v) FCS.

After 48 h, the medium was replaced with one containing approx. 10 % (v/v) FCS and the test compound, or positive control, or solvent and in the presence of metabolic activation additionally 2 % ( v/v) S9-mix.
For the 3 hour treatment time, the medium was replaced by normal medium following two rinses. In the repeat experiment the cells were exposed to the treatment medium without S9-mix for 20 h.

18 h after the start of the treatment, colcemide was added (approx. 0.05μg/ml/culture medium) to the cultures to arrest mitosis and 2 h later (20 h after the start of treatment) metaphase spreads were prepared as follows:

The cultures were made hypotonic by adding about 4 ml of approx. 0.075 M potassium chloride solution at around 37 °C. The cells were then incubated for 20 minutes at approx. 37 °C. The next step was the addition of 1.5 ml fixative.

Then the liquid was replaced by 5 ml fixative (methanol: glacial acetic acid, 3:1). After 10 minutes the procedure was repeated. After at least another 10 minutes, the slides were taken out and airdried for 24 hours. The chromosomes were stained as follows:

staining for 10 minutes in approx. 2 % (w/v) orcein solution
rinsing 3 times in distilled water
rinsing twice in acetone
brief rinsing in acetone/xylene
2 minutes in acetone/xylene
5 minutes in xylene
10 minutes in xylene
embedding in Enteilan® or Corbit®

Duplicate cultures were prepared from each experimental group.

For both treatment times the solvent and the positive controls were prepared 20 h after treatment in the same way.

Evaluation criteria:

Analysis of metaphases
The slides were coded and 25 - 100 metaphases per experimental group and cell culture were examined. The set of chromosomes was examined for completeness and the various chromosomal aberrations were assessed. Only metaphases with 22 ±2 chromosomes are included in the analysis. The metaphases were examined for the following aberrations: chromatid gap, chromosome gap, chromatid break, chromosome break, minute, double minute, chromatid deletion, chromosome deletion, chromatid exchanges including intrachanges, chromosome exchanges including intrachanges, dicentrics, pulverization and ring formation. Furthermore the incidence of polyploid metaphases was determined in 1000 cells of each cell culture.
Additionally a mitotic index was determined by counting the number of cells undergoing mitosis in a total of 1000 cells. The mitotic index is given in per cent.
After the metaphases had been evaluated, the code was broken. The values for the control group were compared with the results from the dose groups and the positive control at each sampling time.

Criteria for a valid assay
The assay was considered valid if the following criteria are met:
the solvent control data were within the laboratory's normal control range for the spontaneous mutant frequency
the positive controls induced increases in the mutation frequency which were both statistically significant and within the laboratory's normal range

Criteria for a positive response
The evaluation of the results was performed as follows:
The test compound is classified as mutagenic if it induces a statistically significant increase in the aberration rate (without gaps) with one or more of the concentrations tested as compared with the solvent controls.
The test compound is classified as mutagenic if there is a concentration-related increase in the aberration rate (without gaps).
The test compound is classified as non-mutagenic if the tests are negative both with and without metabolic activation.

Statistics:

Statistics
The Biometry of the results was performed with a one-sided Fisher - Exact test.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Solubility and preliminary toxicity testing T-9601 was suspended in DMSO.
Evaluation of the solubility of that suspension in cell culture medium showed that 1285μg/ml was the highest practicable concentration and produced precipitate.

Accordingly, the preliminary toxicity study was carried out using a maximum concentration of 1285μg/ml and a range of lower dose levels down to 10μg/ml.

Following treatment for 3 hours, several toxicity was observed at 100μg/ml and above. Survival declined in a dose-related manner reaching 35.3 % in the absence of metabolic activation, respectively 55.4 % in the presence of S9-mix of the solvent control value at a dose level of 750μg/ml.
After 20 hours treatment survival was also dose-dependent reduced reaching 17.5 % of the solvent control value at a concentration of 500μg/ml.
On account of the precipitation of the test substance the extinction values at higher dose levels were slightly increased at both treatment times.

Before treatment, the pH values and osmolality of the treatment media were determined. The addition of test compound solutions did not have any effect on these parameters.

Mutagenicity test
In the main experiments cytotoxicity was also evaluated by treatment of cells seeded in microwell plates. Survival was reduced in a dose-related manner reaching 39.8 % of the solvent control value without S9-mix at the 20 hours treatment time at the highest dose, 250μg/ml and 42.0 % respectively 54.7 % in the absence respectively in the presence of S9-mix at the 3 hours treatment time at a concentration of 750μg/ml.
In accordance to the preliminary experiment the extinction values at the dose level of 1285μg/ml were increased on account of the precipitation of the test substance in the wells.
In the main and in the repeat experiments the mitotic index was reduced (indication of toxicity) after treatment with the highest dose levels.
After treatment with the test compound there was no relevant increase in the number of polyploid cells as compared with the solvent controls.
There was an enhancement of the aberration rates at the 3 h treatment time with 1285μg/ml with S9-mix, but only in one culture. These data were found significantly enhanced in the Fisher's exact-test.
Because of the different results between the duplicate cultures a third independent experiment with two slides and duplicate cultures was performed additionally. In this experiment the enhancement of the aberration rate was not reproduced.

The test compound T-9601 was assessed for its mutagenic potential in vitro in the chromosome aberration test in two independent experiments.
No relevant reproducible enhancement of metaphases with aberrations over the range of the solvent control was found with any of the concentrations used, either with or without metabolic activation by S9-mix. The sensitivity of the test system was demonstrated by the enhanced mutation frequency in the cell cultures treated with the positive control compounds.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

MAIN EXPERIMENT TOXICITY TABLES

 

Table 2a (3 hours treatment time without S9-mix)

	Dose μg/ml	S9-mix	Extinction in microwell plates mean less blank values	Standard deviation	Relative survival*
Solvent control	0	-	0.485	0.03	100.0
		+	0.402	0.03	100.0
T-9601	25	-	0.427	0.01	88.1
		+	0.376	0.03	93.9
	50	-	0.402	0.03	82.9 #
		+	0.338	0.02	84.0 #
	75	-	0.378	0.03	78.0 #
		+	0.340	0.02	84.6 #
	125	-	0.343	0.08	70.6 #
		+	0.308	0.02	78.8 #
	250	-	0.291	0.04	60.0 #
		+	0.277	0.02	69.0 #
	500	-	0.236	0.02	48.6 #§
		+	0.250	0.02	62.2 #§
	750	-	0.204	0.03	42.0 #§
		+	0.220	0.02	54.7 #§
	1285	-	0.318	0.02	65.6 #§
		+	0.268	0.02	66.7 #§

 

Table 2b (20 hours treatment time without S9-mix)

	Doseμg/ml	S9-mix	Extinction in microwell plates mean less blank values	Standard deviation	Relative survival*
Solvent control	0	-	0.489	0.04	100.0
T-9601	5	-	0.480	0.06	98.1
	10	-	0.461	0.02	94.3
	25	-	0.421	0.02	86.0
	50	-	0.378	0.02	77.4 #
	75	-	0.328	0.02	67.1 #
	125	-	0.248	0.03	50.7 #
	250	-	0.195	0.03	39.8 #

 

* = relative survival (mean value/mean value corresponding control x 100)

Solvent control = DMSO

# = microscopical visible precipitation of the test compounds

§= macroscopical visible precipitation of the test compounds

 

TABLE OF THE MITOTIC INDEX

First experiment	Doseμg/ml	S9-Mix	Treatment time (h)	Mitotic index		Mean	Relative mitotic index percent*
Test group				1	2
Solvent control DMSO	0.0	-	3	5.1	5.7	5.9	100.0
T-9601	125.0	-	3	4.5	4.3	4.4	74.8
T-9601	500.0	-	3	2.8	4.3	3.6	61.0
T-9601	1285.0	-	3	4.6	3.2	3.9	66.1
Positive control EMS	1500.0	-	3	5.7	3.3	4.5	76.3
Solvent control DMSO	0.0	+	3	7.0	8.2	7.6	100.0
T-9601	125.0	+	3	7.3	7.2	7.3	96.1
T-9601	500.0	+	3	5.4	6.5	6.0	79.0
T-9601	1285.0	+	3	4.3	5.0	4.7	61.8
Positive control CPA	3.0	+	3	4.1	3.8	4.0	52.6
Second experiment	Doseμg/ml	S9-mix	Treatment time (h)	Mitotic index		Mean	Relative mitotic index percent*
Test group				1	2
Solvent control DMSO	0.0	-	20	5.0	5.4	5.2	100.0
T-9601	25.0	-	20	5.6	4.2	4.9	94.2
T-9601	75.0	-	20	4.9	4.7	4.8	92.3
T-9601	250.0	-	20	2.3	2.9	2.6	50.0
Positive control EMS	400.0	-	20	3.1	2.9	3.0	57.7
Third experiment	Doseμg/ml	S9-mix	Treatment time (h)	Mitotic index		Mean	Relative mitotic index percent*
Test group				1	2
Solvent control DMSO	0.0	+	3	8.4	9.1	8.8	100.0
T-9601	1285.0	+	3	4.0	6.6
				4.8	5.2	5.2	59.1
Positive control CPA	3.0	+	3	5.4	3.2	4.3	48.9

* The mitotic index was determined in 1000 cells

 

TABLE OF THE NUMBER OF POLYPLOID CELLS

First experiment	Doseμg//ml	S9-mix	Treatment time (h)	Polyploid cells*		Mean
Test group				1	2
Solvent control DMSO	0.0	-	3	2	1	1.5
T-9601	125.0	-	3	0	3	1.5
T-9601	500.0	-	3	0	2	1.0
T-9601	1285.0	-	3	0	0	0.0
Positive control EMS	1500.0	-	3	1	1	1.0
Solvent control DMSO	0.0	+	3	4	1	2.5
T-9601	125.0	+	3	0	1	0.5
T-9601	500.0	+	3	2	0	1.0
T-9601	1285.0	+	3	0	4	2.0
Positive control CPA	3.0	+	3	0	1	0.5
Second experiment	Doseμg/ml	S9-mix	Treatment time (h)	Polyploid cells*		Mean
Test group				1	2
Solvent control DMSO	0.0	-	20	0	4	2.0
T-9601	25.0	-	20	6	6	6.0
T-9601	75.0	-	20	66	2	4.0
T-9601	250.0	-	20	0	1	0.5
Positive control EMS	400.0	-	20	0	1	0.5
Third experiment	Doseμg/ml	S9-mix	Treatment time (h)	Polyploid cells*		Mean
Test group				1	2
Solvent control DMSO	0.0	+	3	3	2	2.5
T-9601	1285.0	+	3	1	7
				5	6	4.8
Positive control CPA	3.0	+	3	2	0	1.0

* The number of polyploid cells was determined in 1000 cells

Conclusions:

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

T-9601 was not mutagenic in this chromosome aberration test system in vitro with cells of the V79 Chinese hamster cell line under the conditions described in this report.

Executive summary:

The present study was conducted in compliance with OECD Guideline For Testing Of Chemicals, 473 "Genetic Toxicology:In Vitro Mammalian Chromosome Aberration Test". Adopted: July 21st, 1997 and U.S. EPA: OPPTS 870.5375 Health Effects Test Guidelines In Vitro Mammalian Chromosome Aberration Test, August 1998 and EEC Directive 92/69, L 383 A, Annex B. 10, p. 148 -150. This study was conducted in compliance with the Principles of Good Laboratory Practice (GLP).

 

In this study the potential of T-9601to induce chromosome aberrations was investigated in V 79 cells of the Chinese hamster lung in vitro. For each experiment duplicate cultures were used for each concentration.

 

The test compound was suspended in DMSO and tested at the following concentrations: First experiment with 3 h treatment time:

without S9-mix: 25, 50, 75,125*, 250, 500*, 750 and 1285*μg/ml

with S9-mix:   25, 50, 75, 125*, 250, 500*, 750 and 1285*μg/ml

Second experiment with 20 h treatment time:

without S9-mix: 5, 10, 25*, 50, 75*. 125 and 250*μg/ml

Third experiment with 3 h treatment time:

with S9-mix: 1285*μg/ml

*= evaluated slides

 

The concentration ranges were based on the results of preliminary testing for solubility and toxicity. The highest concentration produced a distinct lowering of the mitotic index.

 

At concentrations of 500μg/ml and above macroscopic visible precipitation of the test substance on-to the slides was observed. Microscopically precipitation of the test compound was observed at 50μg/ml and above.

 

There was an enhancement of the aberration rates at the 3 h treatment time with 1285μg/ml with S9-mix, but only in one culture. These data were found significantly enhanced in the Fisher's exact-test.

 

Because of the different results between the duplicate cultures a third independent experiment with two slides and duplicate cultures was performed. In this experiment the enhancement of the aberration rate was not reproduced.

 

Without S9-mix up to the highest investigated dose the test compound induced no significant increase in the number of chromosome aberrations.

 

Appropriate reference mutagens used as positive controls showed a significant increase in chromosome aberrations, thus indicating the sensitivity of the assay, and the efficacy of the S9-mix.

 

In conclusion, T-9601does not induce chromosome mutations (=aberrations) in V79 Chinese hamster cells, both in the presence as well as in the absence of a metabolic activation system, under the experimental conditions described.

 

T-9601 is therefore considered to be non-mutagenic in this chromosome aberration assay.

Reference 3

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:

09 November 1998 to 14 December 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: In vitro Mammalian Cell Gene Mutation Test EEC Directive 87/302, L133, pp. 61 - 63, March 1987

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell transformation assay

Target gene:

HPRT (hypoxanthine-guanine phosphoribosyl transferase) locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Cell culture medium: MEM (minimal essential medium) with Hanks-salts and 25 mM Hepes-buffer

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

The compound was suspended in DMSO and tested at the following concentrations:
without S9-mix; 5.0, 7.5, 12.5, 25.0, 50.0, 75.0. 125.0, 250.0, 500.0, 750.0 and 1285.0 μg/ml (main mutation experiment)
50.0, 75.0, 125.0, 250.0, 500.0, 750.0, 1000.0 and 1285.0 μg/ml (repeat mutation experiment)
with S9-mix: 5.0, 7.5, 12.5, 25.0, 50.0, 75.0, 125.0, 250.0, 500.0, 750.0 and 1285.0 μg/ml (main mutation experiment)
50.0, 75.0, 125.0, 250.0, 500.0, 750.0, 1000.0 and 1285.0 μg/ml (repeat mutation experiment)

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: without metabolic activation: EMS (Ethyl methane sulfonate), with metabolic activation: DMBA (9,10-dimethyl-1 ,2-benzanthracene)

Details on test system and experimental conditions:

Reference Compounds

Without metabolic activation
Name or number of compound (I.N.N. orU.SAN): ethyl methane sulfonate
Synonyms: EMS
Formula of the compound: C3Ha03S
CAS-Register number: 62-50-0
Product number / Code: 820774
Supplier of reference compound: Dr. Theodor Schuchardt & Co. Chemische Fabrik, Germany
Batch number: 40606721
Certificate of analysis: certificated by the supplier, Analytical Department, Dr. Bolkart dated December 18th, 1995

With metabolic activation
Name or number of compound (I.N.N. orU.S.A.N): 9,10-dimethyl-1,2-benzanthracene
Synonyms: DMBA
Chemical name: 7,12-dimethylbenz[a]anthracene
CAS-Register number: 57-97-6
Product number / Code: 21,626-7
Supplier of reference compound: Fa. Aldhch – Chemie, Germany
Batch number: TL 1825LL
Certificate of analysis: certificated by the supplier, Aldrich chemical company, David Swessel, dated April 3rd, 1996

Test system

Test groups
with and without metabolic activation: 5.0, 7.5, 12.5, 25.0, 50.0, 75.0, 125.0, 250.0, 500.0, 750.0 and 1285.0 μg/ml (main mutation experiment)
50.0, 75.0, 125.0, 250.0, 500.0, 750.0, 1000.0 and 1285.0 μg/ml (repeat mutation experiment)

Control groups
negative controls: untreated control & cultures treated with the solvent

positive controls:
without metabolic activation: EMS (Ethyl methane sulfonate)
with metabolic activation: DMBA (9,10-dimethyl-1,2-benzanthracene)

Formulation of test compounds: suspended in DMSO at appropriate concentrations immediately before use.

Formulation of reference compounds: EMS dissolved in cell culture medium on the day of treatment, final concentration: 1.0 mg/ml = 8 mM.
DMBA dissolved in DMSO and frozen in small portions. Aliquot thawed on the day of treatment, final concentration in cell culture medium: 7.7 μg/ml = 30 μM

Source of biological material: cell bank of "Genetic Toxicology", HMR Deutschland GmbH, ProTox

Test organism: cell line V79 of Chinese hamster lung fibroblasts

Cell culture medium: MEM (minimal essential medium) with Hanks-salts and 25 mM Hepes-buffer

Experimental conditions in vitro: approx. 37 °C and approx. 4 % CO2 in plastic flasks
Observations and Measurements

Preparation and storage of a liver homogenate fraction (S9)
The S9 fraction was prepared by the testing facility according to Ames et. al (1975). Male Sprague Dawley rats (200-300 g), supplied by Harlan Winkelmann, Gartenstrasse 27, 33178 Borchen, Germany, received a single intraperitoneal injection of Aroclor 1254 (500 mg/kg body weight) 5 days before killing. The livers were removed from at least 5-6 animals at approx. 0 to 4 °C using cold sterile solutions and glassware, and were then pooled and washed in approx. 150 mM KCI (approximately 1 ml/g wet liver). The washed livers were cut into small pieces and homogenized in three volumes of KCI. The homogenate was cenfrifuged at approx. 9000g for 10 minutes. The supernatant, the S9 fraction, was divided into small portions, rapidly frozen and stored at approx. - 80 °C for not longer than six months. The protein content was determined for every batch. Also for every batch of S9 an independent validation was performed with a minimum of two different mutagens, e.g., 2-aminoanthracene and dimethylbenzanthracene to confirm metabolic activation by microsomal enzymes.

Preparation of S9-mix
Sufficient S9 fraction was thawed to room temperature immediately before each test. An appropriate quantity of S9 fraction (batch no. 98/1 for both mutation experiments, protein concentration 51.8 g/l) was mixed with S9 cofactor solution to yield a final protein concentration of 0.3 mg/ml in the cultures which was kept on ice until used. This preparation is termed S9-mix. The concentrations of the different components of the S9-mix were:
8 mM MgCI3
33 mM KCI
5 mM glucose-6-phosphate
5 mM NADP
100 mM phosphate buffer pH 7.4

Cell culture
Large stocks of the mycoplasma-free V79 cell line are stored in liquid nitrogen in the cell bank of "Genetic Toxicology", thus permitting repeated use of the same cell culture batch for numerous experiments. The identical characteristics of the cells ensure comparability of the experimental parameters.
Thawed stock cultures were kept at approx. 37 °C and approx. 4 % C03 in 175 cm2 plastic flasks. About 5 x 105 to 1 x 106 cells were seeded into each flask in 30 ml of MEM-medium supplement with approx. 10 % (v/v) FCS (fetal calf serum) containing approx. 2 mM L-glutamine and approx. 0.1 % (w/v) neomycinsulfate. The cells were subcultured twice a week.
For the selection of mutants the medium was supplemented with approx. 11 μg/ml thioguanine.

Toxicity experiments and dose range finding
A preliminary toxicity test was undertaken in order to select appropriate dose levels for the mutation assay. In this test a wide range of dose levels of test compound was used. Cell cultures were subjected to the same treatment conditions as in mutation assays, and the survival of the cells was subsequently determined.

The test included the following treatments:
Solvent control : the maximum final concentration of organic solvents will not exceed approx. 1 % (v/v).
Test compound : the highest dose level for the preliminary toxicity test was determined by the solubility of the test compound up to the maximum of 10 mM or 5000 μg/ml.
Treatments were performed both in the presence and absence of S9 metabolic activation system using a single cell culture at each test point.

Test procedure
In preliminary toxicity experiments approximately 4500 cells were seeded in each well of a microliter plate, allowed to attach overnight and then exposed to the test and control compound for four hours.
For each concentration at least 6 wells were used. Approx. 24 hours after treatment, the cells were fixed and stained with crystal violet.
Survival was determined by measurement of the crystal violet extinction.

In the main mutation experiments the cultures for assessing toxicity were prepared and treated with the test compound in the same way as for the preliminary experiment. 24 hours after seeding of approx. 4500 cells per well in a microtiter plate, the medium was replaced with serum-reduced (5 % v/v) medium containing the test compound to which either buffer or S9-mix was added as appropriate. After 4 hours the treatment medium was replaced with normal medium after rinsing twice with this. The cultures were stained with crystal violet and survival was determined after an incubation period of approx. 24 hours.

Rationale for dose selection
For non-toxic, freely soluble test compounds, the top dose should be 10 mM or 5000 ug/ml according to international testing guidelines.
For non-toxic, poorly soluble test compounds, the top dose should be the highest evaluable dose.
For toxic compounds the percentage survival relative to the solvent control should be calculated for each treatment. The dose level which resulted in a predicted survival of less than 30 % should be chosen as the highest dose level. At least eight respectively seven lower dose levels should be also included in each experiment.

Mutagenicity test
Two independent mutation tests were performed.
Two-day old, exponentially growing cultures which were more than 50 % confluent were trypsinated and a single cell suspension was prepared. The trypsin concentration was approx. 0.25 % (v/v) in Ca-Mg-free salt solution. The Ca-Mg-free salt solution was prepared as follows (per liter): NaCI 6.8 g; Ka 0.4 g; glucose 1 g; NaHC03 2.2 g; phenol red 5 mg; trypsin 2.5 g.
Subsequently the cells were replated to determine the mutation frequency and plating efficiency.

The treatment schedule of the mutagenicity test is described below:

Day 1: Subculturing of an exponentially growing culture
a) Approx. 4500 cells in each well of a microtiter plate for determination of the plating efficiency.
b) 6x 105 - 1 x 106 cells in 175 cm2 flasks with 30 ml medium for the mutagenicity test, one flask per experimental point.

Day 2: Treatment of a) and b) with the test compound in the presence and absence of S9-mix (final protein concentration: approx. 0.3 mg/ml) for 4 hours.

Day 3: Fixation and staining of the cells in a) for the determination of the plating efficiency.

Day 5: Subculturing of b) in 175 cm2 flasks

Day 9: Subculturing of b) in five 75 cm1 flasks with culture medium containing 6-thioguanine:
Mutant selection (about 300 000 cells/flask);
subculturing of b) in two 25 cm2 flasks for plating efficiency (about 400 cells per flask)

Day 16: Fixation and staining of colonies of b) - from subcultures seeded on day 9.

All incubations were carried out at approx. 37 °C and 4 % CO2.
Staining was performed with approx. 10 % (v/v) methylene blue in approx. 0.01 % (w/v) KOH solution.
Only colonies with more than 50 cells were counted.

Evaluation criteria:

Evaluation of data

Criteria for a valid assay
The assay is considered valid if the following criteria are met:
the solvent control data are within the laboratory's normal control range for the spontaneous mutant frequency
the positive controls induced increases in the mutation frequency which were both statistically significant and within the laboratory's normal range
the plating efficacy for the solvent control was greater than 50 %

Criteria for a positive response
The test compound is classified as mutagenic if:
it reproducibly induces with one of the test compound concentrations a mutation frequency that is three times higher than the spontaneous mutant frequency in this experiment
there is a reproducible dose-related increase in the mutation frequency. Such an evaluation may be considered independently from the number induced mutants
survival of the responding dose group is at least 30 %
However, in a case by case evaluation both decisions depend on the level of the corresponding negative control data.

Statistics:

The biometry of the results for the test compound is performed off-line with the MANN-WHITNEY-U-TEST

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Solubility and toxicity
T-9601 was suspended in DMSO.
Evaluation of the solubility of that suspension in cell culture medium showed that 1285.0 μg/ml was the highest practicable concentration and produced precipitate.
Accordingly, the preliminary toxicity study was carried out using a maximum concentration of 1285.0 μg/ml and a range of lower dose levels down to 10.0 pg/ml.
Following treatment in the absence of S9 metabolic activation, several toxicity was observed. Survival declined in a dose-related manner reaching 23.0 % of the solvent control value at the concentration of 750 μg/ml.
In the presence of S9 metabolic activation survival declined steeply up to the concentration of 500.0 μg/ml where survival was reduced to 64.8 % of the solvent control value.
Based on these results 1285 μg/ml was selected as the maximum dose level for the main mutation experiments in both the absence and in the presence of S9-mix. Ten lower concentrations down to 5 μg/ml were included in the main mutation experiment and seven lower concentrations down to 50 μg/ml in the repeat mutation experiment.

Mutagenicity
Experimental design
Two independent mutation assays to examine resistance to 6-thioguanine were performed.
In the absence and in the presence of S9 metabolic activation dose levels of 5.0, 7.5, 12.5, 25.0, 50.0, 75.0,125.0, 250.0, 500.0, 750.0 and 1285.0 μg/ml were used in the main mutation experiment, For the repeat mutation experiment a dose range of 50.0, 75.0, 125.0, 250.0, 500.0, 750.0, 1000.0 and 1285.0 μg/ml was selected in the absence and in the presence of S9-mix.
Before treatment, the pH values and osmolality of the treatment media were determined. The addition of test compound solutions did not have any effect on these parameters.

Survival after treatment
In the absence of S9 metabolic activation in both mutation experiments a dose-related decrease in survival was observed reaching 53.2 % respectively 39.4 % of the solvent control value in the microtiter plates at the highest dose level tested, 1285.0 μg/ml.
In the presence of S9 metabolic activation survival decreased in a dose-related manner reaching approximately 45.3 % respectively 53.8 % of the solvent control value in the microtiter plates after treatment at the highest dose level, 1285.0 μg/ml.

Mutation results
The test compound T-9601 was assessed for its mutagenic potential in vitro in the HPRT-test in two independent experiments without metabolic activation and two independent experiments with metabolic activation.
In the presence of metabolic activation a statistically significant increase of the mutation frequency was observed only at a concentration of 7.5 μg/ml. This effect was not dose-dependent and not three fold higher than the corresponding controls and therefore of no biological relevance.
No relevant reproducible increases in the mutant colonies or mutant frequency over the range of the solvent control was found with any of the concentrations used, either with or without metabolic activation by S9-mix.
The sensitivity of the test system and efficacy of the S9-mix was demonstrated by the enhanced mutation frequency in the cell cultures treated with the positive control compounds.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Toxicity Data (Main mutation experiment)

	Dose μg/ml	S9-mix	Extinction in microwell plates mean less blank values	Standard deviation	Relative survival*
Negative control	0.0	-	0.572	0.02	121.5
		+	0.451	0.05	125.3
Solvent control	0.0	-	0.471	0.04	100.0
		-	0.424	0.06	100.0
		+	0.360	0.06	100.0
		+	0.411	0.08	100.0
Positive control	1000.0	-	0.504	0.01	107.0
	7.7	+	0.361	0.04	100.3
T-9601	5.0	-	0.499	0.02	105.9
		+	0.461	0.03	128.0
	7.5	-	0.529	0.08	112.3
		+	0.462	0.03	128.3
	12.5	-	0.495	0.02	105.0
		+	0.448	0.04	124.4
	25.0	-	0.440	0.01	93.5
		+	0.437	0.03	121.4
	50.0	-	0.420	0.02	89.1 #
		+	0.378	0.03	105.2 #
	75.0	-	0.398	0.02	84.1 #
		+	0.413	0.03	114.7 #
	125.0	-	0.483	0.10	113.9 #
		+	0.387	0.03	94.2 #
	250.0	-	0.319	0.02	75.2 #§
		+	0.418	0.05	101.8 #§
	500.0	-	0.253	0.02	59.6 #§
		+	0.325	0.05	79.1 #§
	750.0	-	0.276	0.02	65.2 #§
		+	0.256	0.05	62.3 #§
	1285.0	-	0.226	0.03	63.2 #§
		+	0.186	0.04	45.3 #§

# microscopical visible precipitation

§ macroscopical visible precipitation

* relative survival (mean value / mean value corresponding control x 100)

Solvent = DMSO

Positive control without S9-mix = EMS

Positive control with S9-mix = DMBA

 

Mutagenicity Data – Part 1 (Main experiment)

	Dose μg/ml	S9-mix	Number of cells per flask				Factor* calculated	Cells* seeded	Cells*** survived
			Seeded	Found		Mean
			I / II	I	II
Negative control	0.0	-	403	158.0	164.5	161.3	0.40	342900	137203
		+	401	101.0	98.0	99.5	0.25	337500	83744
Solvent control 1 (DMSO)	0.0	-	398	370.5	370.0	370.3	0.93	292350	271966
		+	399	370.0	372.0	371.0	0.93	298800	277832
Solvent control 2 (DMSO)	0.0	-	400	313.0	323.5	318.3	0.80	291150	231646
		+	402	201.5	200.5	201.0	0.50	287700	143850
Positive control (EMS)	1000.0	-	400	288.0	273.5	280.8	0.70	285000	200034
Positive control (DMBA)	7.7	+	398	281.0	299.0	290.0	0.73	276750	201652
T-9601	5.0	-	399	258.5	261.0	259.8	0.65	291900	190028
		+	400	368.0	386.5	377.3	0.94	258600	243892
	7.5	-	401	188.5	191.5	190.0	0.47	291000	137880
		+	401	284.0	274.0	279.0	0.70	326400	227096
	12.5	-	399	278.0	277.0	277.5	0.70	307800	214071
		+	394	271.0	278.5	274.8	0.70	331650	231271
	25.0	-	399	348.0	362.5	355.3	0.89	294300	262030
		+	402	361.5	380.0	370.8	0.92	276150	254683
	50.0	-	403	320.0	310.5	315.3	0.78	288150	225408
		+	402	292.5	296.0	294.3	0.73	280650	205426
	75.0	-	399	357.0	338.5	347.8	0.87	294300	256498
		+	405	276.5	272.5	274.5	0.68	299250	202825
	125.0	-	400	381.0	338.0	359.5	0.90	294300	264502
		+	399	340.0	351.0	345.5	0.87	269400	233277
	250.0	-	402	368.0	353.0	360.5	0.90	288750	258941
		+	401	450.5	449.0	449.8	1.12	258150	289534
	500.0	-	403	322.0	314.0	318.0	0.79	279750	220748
		+	402	302.0	314.5	308.3	0.77	288900	221526
	750.0	-	402	271.0	299.5	285.3	0.71	349200	247784
		+	399	313.0	314.0	313.5	0.79	336000	264000
	1285.0	-	400	156.0	144.0	150.0	0.38	337500	126563
		+	405	301.0	306.5	303.8	0.75	347250	260438

* Factor calculated: mean value / number of cells per flask seeded

** Cells seeded in 6-thioguanine (TG) containing medium

*** Cells survived after plating in (TG) containing medium (cells seeded x factor calculated)

 

Mutagenicity Data – Part 2 (Main experiment)

	Dose μg/ml	S9-mix	Number of mutant colonies						Standard deviation	Mutation frequency	Stat. sig.
			I	II	III	IV	V	Mean
Negative control	0.0	-	1	4	1	3	2	2.2	1.30	16.0
		+	2	1	2	3	0	1.6	1.14	19.1
Solvent control 1 (DMSO)	0.0	-	3	3	1	2	1	2.0	1.00	7.4
		+	1	1	1	1	3	1.4	0.89	5.0
Solvent control 2 (DMSO)	0.0	-	9	11	6	3	6	7.0	3.08	30.2
		+	3	1	2	7	2	3.0	2.35	20.9
Positive control (EMS)	1000.0	-	166	147	161	156	148	155.8	8.20	777.9	*
Positive control (DMBA)	7.7	+	35	35	45	40	36	38.2	4.32	189.4	*
T-9601	5.0	-	1	0	0	1	3	1.0	1.22	5.3
		+	5	3	1	1	1	2.2	1.79	9.0
	7.5	-	1	1	1	2	0	1.0	0.71	7.3
		+	8	5	12	10	6	8.2	2.88	35.1	*
	12.5	-	3	3	0	4	3	2.6	1.52	12.1
		+	3	2	1	3	1	2.0	1.00	8.6
	25.0	-	5	5	1	2	3	3.2	1.79	12.2
		+	1	3	1	4	4	2.6	1.52	10.2
	50.0	-	0	0	4	1	1	1.2	1.64	5.3
		+	1	1	1	3	0	1.2	1.10	5.8
	75.0	-	1	4	5	5	2	3.4	1.82	13.3
		+	5	1	2	7	8	4.6	3.05	22.7
	125.0	-	1	4	2	0	2	1.8	1.48	6.8
		+	2	1	2	7	3	3.0	2.35	12.9
	250.0	-	5	6	2	4	2	3.8	1.79	14.7
		+	5	0	2	2	4	2.6	1.95	9.0
	500.0	-	4	6	2	1	2	3.0	2.00	13.6
		+	4	1	8	3	5	4.2	2.59	19.0
	750.0	-	6	7	3	5	3	4.8	1.79	19.4
		+	11	8	2	7	11	7.8	3.70	29.5
	1285.0	-	1	6	5	5	3	4.0	2.00	31.6
		+	1	7	2	2	3	3.0	2.35	11.5

Mutation frequency (mutant colonies per 1 million cells); mean value / cells surviving

* Statistical significant (p≤0.05) Mann-Whitney-U-Test

Conclusions:

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

T-9601 did not induce gene mutation, i.e. was not mutagenic, in this HPRT-test with V79 Chinese hamster cells, either in the presence or in the absence of metabolic activation.

Executive summary:

The present study was conducted in compliance with OECD Guideline For Testing Of Chemicals, 476 "Genetic Toxicology: In vitro Mammalian Cell Gene Mutation Test". Adopted: July 21, 1997 and U.S. Environmental Protection Agency (EPA) Health Effect Test Guidelines, OPPTS 870.5300, In Vitro Mammalian Cell Gene Mutation Test, august 1998 and In vitro Mammalian Cell Gene Mutation Test EEC Directive 87/302, L133, pp. 61 - 63, March 1987. This study was conducted in compliance with the Principles of Good Laboratory Practice (GLP).

 

The study was performed to investigate the potential of T-9601 to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster in vitro.

Two independent experiments were conducted both with and without an exogenous rat liver microsomal activation system (S9-mix).

The compound was suspended in DMSO and tested at the following concentrations:

without S9-mix; 5.0, 7.5, 12.5, 25.0, 50.0, 75.0. 125.0, 250.0, 500.0, 750.0 and1285.0 μg/ml (main mutation experiment)

50.0, 75.0, 125.0, 250.0, 500.0, 750.0, 1000.0 and 1285.0 μg/ml (repeat mutation experiment)

With S9 -mix: 5.0, 7.5, 12.5, 25.0, 50.0, 75.0, 125.0, 250.0, 500.0, 750.0 and 1285.0 μg/ml (main mutaation experiment)

50.0, 75.0, 125.0, 250.0, 500.0, 750.0, 1000.0 and 1285.0 μg/ml (repeat mutation experiment)

 

The concentration ranges were based on the results of preliminary tests for solubility and toxicity. The highest concentration showed slight toxic effects with and several toxic effects without metabolic activation.

In the presence of metabolic activation a significant increase of the mutation frequency was observed only at a concentration of 7.5 μg/ml. This effect was not dose-dependent and not three fold higher than the corresponding controls and therefore of no biological relevance.

Up to the highest investigated dose no further increase in mutant colony numbers was obtained in two independent experiments.

Appropriate reference mutagens used as positive controls showed a distinct increase in induced mutant colonies, thus indicating the sensitivity of the assay, and the efficacy of the S9-mix.

 

In conclusion, the test substance does not induce gene mutations in the HPRT-test with V79 Chinese hamster cells, both in the presence as well as in the absence of a metabolic activation system under the experimental conditions described.

The test substance is therefore considered to be non-mutagenic in this HPRT assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The source substance was negative in the following tests:

1) Chromosome Aberration Assay – V79 Chinese Hamster Lung

2) HPRT Assay – V79 Chinese Hamster Lung

 

The source substance was positive in the following test:

3) Ames Test – Salmonella and E. coli

 

Two separate gene mutation tests in mammalian cells demonstrated that the source substance does not have any mutagenic properties in V79 Chinese Hamster Lung cells. In the bacterial mutation test strong positive effects were obtained with and without S-9 mix at doses which induced precipitations. The results lead to the conclusion that the susbstance is mutagenic in the bacterial systems with and without an exogenous metoblizing system. However it should be noted that the specific method according to Privall - Mitchell for azo dyes was not utilised in this study; hence the results may be due to redundancies within the method employed. This statement is further enforced by the negative mutagenicity results observed in the other in vitro studies conducted.

Repeat-dose studies with oral treatment in rats did not reveal any tumorigenic properties which could be related to the administration of the test substance. Consequently, additional testing of the test substance in an in vitro gene mutation study in mammalian cells does not appear scientifically necessary and this test has been waived. Due to the structural similarity, this results is assumed appropriate for the target substance, too.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The above studies have all been ranked reliability 1 according to the Klimisch et al system. This ranking was deemed appropriate because the studies were conducted to GLP an in compliance with agreed protocols. Sufficient dose ranges and numbers are detailed; hence it is appropriate for use based on reliability and animal welfare grounds. As the effects are considered adaptive rather than toxicological, no classification is proposed. This result is also assumed for the target substance.

The above results triggered no classification under the CLP Regulation (EC No 1272/2008). No classification for prolonged effects is therefore required.