tetrasodium 2-(4-fluoro-6-(methyl-(2-(sulfatoethylsulfonyl)ethyl)amino)-1,3,5-triazin-2-ylamino)-5-hydroxy-6-(4-methyl-2-sulfonatophenylazo)naphthalene-1,7-disulfonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance was found to be mutagenic in the bacterial reverse mutation assay but not in the in vitro mammalian cell gene mutation assay or in in vivo mouse micronucleus testing.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From September 6, 1999 to November 05, 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - 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 gene mutation test using the Hprt and xprt genes

Target gene:

HPRT (i.e., hypoxanthine-guanine phosphoribosyl transferase) locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

-Source of cells: Cell bank of "Genetic Toxicology", Aventis Pharma Deutschland GmbH, ProTox
-Cell culture medium: MEM (minimal essential medium) with Hankssalts and 25 mM Hepes-buffer
-Experimental conditions in vitro: Approximately 37°C and approximately 4% CO2 in plastic flasks

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from rat liver

Test concentrations with justification for top dose:

Without S9-mix: 50, 100, 250, 500, 750, 1,000, 1,500, 2,000, 2,500* and 3,000*# µg/mL

With S9-mix: 50, 100, 250, 500, 750, 1,000, 1,500, 2,000, 2,500* and 3,000*# µg/mL

# = only in the repeat main experiment tested concentration
* = because of high cytotoxicity in the repeat main experiment no mutant selection was performed

Vehicle / solvent:

MEM Hank's cell culture medium

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

(without metabolic activation)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9,10-dimethylbenzanthracene

Remarks:

(with metabolic activation)

Details on test system and experimental conditions:

Mutagenicity test
Two independent mutation tests were performed. Exponentially growing cultures which were more than 50% confluent were trypsinated by an approximately 0.25% (v/v) trypsin ready for use (mfr. Gibco). A single cell suspension was prepared. Subsequently the cells were replated to determine the mutation frequencyand plating efficiency.

Test procedure
In the main mutation experiments the cultures for assessing toxicity were prepared and treated with the test substance in the same way as for the preliminary experiment (i.e., described below in any other information on materials and methods incl. tables section). 24 h after seeding of approximately 4,500 cells per well in a microtiter plate, the medium was replaced with serum-reduced (i.e., 5%v/v) medium containing the test substance to which either buffer or S9-mix was added as appropriate. After 4 h the treatment medium was removed and the cells were rinsed twice with normal medium. Thereafter normal medium was added to the wells. The cultures were stained with crystal violet and survival was determined after an incubation period of approximately 24 h.


The treatment schedule of the mutagenicity test is described below

Day 1: Subculturing of an exponentially growing culture

a) Approximately 4,500 cells in each well of a microtiter plate for determination of the plating efficiency.

b) 6 X 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 substance in the presence and absence of S9-mix (final protein concentration: approximately 0.3 mg/mL) for 4 h.

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

Day 5 or 6: Subculturing of b) in 175 cm2 flasks.

Day 9: Subculturing of b) in five 75 cm2 flasks with culture medium containing 6-thioguanine: Mutant selection (i.e., about 3,00,000 cells/flask); subculturing of b) in two 25 cm2 flasks for plating efficiency (i.e., 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 approximately 37°C and 4% CO2, Staining was performed with approximately 10% (v/v) methylene blue in approximately 0.01% (w/v) KOH solution. Only colonies with more than 50 cells were counted.

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

- The plating efficacy for the solvent control was greater than 50%

Criteria for a positive response

The test substance is classified as mutagenic if:

- It reproducibly induces with one of the test substance concentrations a mutation frequency that is three times higher than the spontaneous mutant frequency in this experiment.

- There is a reproducible concentration-related increase in the mutation frequency. Such an evaluation may be considered independently from the enhancement factor for 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 substance is performed off-line with the Mannwhitney- U-Test.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(>= 500 µg/mL (with and without metabolic activation)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- The test substance 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.
- No relevant reproducible increases in the mutant colonies or mutant frequency three times 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 substances.

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 50, 100, 250, 500, 750, 1,000, 1500, 2,000 and 2,500 µg/mL were selected for the main mutation experiment and dose levels of 100, 250, 500, 750, 1,000, 1,500, 2,000, 2,500 and 3,000 µg/mL for the repeat mutation assay. Because of high cytotoxicity with the concentrations of 2,500 and 3,000 µg/mL in the repeat test no mutant selection was performed.

 

-Before treatment, the pH values and osmolality of the treatment media were determined. The addition of test substance 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 26.2%, respectively 30.7% of the solvent control value in the microtiter plates at the highest dose level tested, 2,500 µg/mL, respectively 3,000 µg/mL.

 

- In the presence of S9 metabolic activation survival decreased in a dose-related manner reaching approximately 49.8%, respectively 46.7% of the solvent control value in the microtiter plates after treatment at the highest dose level, 2,500 µg/mL, respectively 3,000 µg/mL.

Solubility and toxicity

 

-Test substance was dissolved in MEM Hank's cell culture medium. Evaluation of the solubility showed that 5,000 µg/mL was the highest practicable concentration and produced no precipitate.

 

- Accordingly, the preliminary toxicity study was carried out using a maximum concentration of 5,000 µg/mL and a range of lower dose levels down to 10 µg/mL.

 

- In the absence and in the presence of S9 metabolic activation survival declined steeply between the three highest concentrations. At a concentration of 1,000 µg/mL survival was reduced to 58.1%,respectively 70.9%of the solvent control value, while at a dose level of 5,000 µg/mL only 3.4%,respectively 2.4%of the solvent control survived.

 

-Based on these results 2,500 µg/mL was selected as the maximum dose level for the main mutation experiment and 3,000 µg/mL for the repeat mutation experiment in both the absence and in the presence of S9-mix. Eight lower concentrations down to 50 µg/mL, respectively 100 µg/mL were also included.

Conclusions:

The test substance was found to be non-mutagenic in the HPRT assay.

Executive summary:

A study was conducted to investigate the potential of the test substance to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster lung according to OECD Guideline 476, EU Method B.17 and EPA OPPTS 870.5300 Method, in compliance with GLP.

 

Two independent experiments were conducted both with and without metabolic activation (i.e., S9 mix). The substance was dissolved in cell culture medium and tested at the concentrations ranging from 50-3,000 µg/mL with and without metabolic activation, based on the results of preliminary testing for solubility and toxicity.

 

Positive controls showed a significant increase in induced mutant colonies, thus indicating the sensitivity of the assay and the efficacy of the S9-mix. The test substance did not induce a relevant reproducible increase in the mutant colonies or mutant frequency three times over the range of the solvent control up to the highest investigated dose in two independent experiments, either with or without metabolic activation by S9-mix.

 

Under the study conditions, the test substance was found to be non-mutagenic in the HPRT assay.

Reference 2

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:

From August 11, 1999 to August 19, 1999

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:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Concentration of stock solution: 50 mg/mL

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

First plate incorporation test
a: without metabolic activation:
50, 160, 500, 1,600 and 5,000 µg/plate
b: with metabolic activation:
50, 160, 500, 1,600 and 5,000 µg/plate

Second plate incorporation test:
a: without metabolic activation:
500, 1,600, 2,000, 3,000, 4,000 and 5,000 µg/plate (TA 1535)
b: with metabolic activation:
500, 1,600, 2,000, 3,000, 4,000 and 5,000 µg/plate (TA 1535)

Vehicle / solvent:

Deionized water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without metabolic activation for strain TA 100 and TA 1535

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without metabolic activation for strain TA 1537

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without metabolic activation for strain TA 98

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without metabolic activation for strain WP2uvrA

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation (i.e., S9-mix from rat liver) for all strains

Details on test system and experimental conditions:

Assay procedure

Each 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 highest concentration in the first mutation experiment was usually 50 mg/mL of the test substance in the chosen solvent, which provided a final concentration of 5,000 µg/plate. Further dilutions of 1,600, 500, 160 and 50 µg/plate were used. Suitable dose levels used in the second experiment may be different depending on any toxicity seen in the first experiment. A reduction in the number of spontaneously occurring colonies and visible thinning of the bacterial lawn were used as toxicity indicators. Thinning of the bacterial lawn was evaluated microscopically.

In both tests top agar was prepared which, for the Salmonella strains, contained 100 mL agar (i.e., 0.6% (w/v) agar, 0.5% (w/v) NaCI) with 10 mL of a 0.5 mM histidine-biotin solution. For E. coli histidine was replaced by tryptophan (i.e., 2.5 mL, 0.5 mM). The following ingredients were added (in the following order) to 2 mL of molten top agar at approximately 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 substance solution (i.e., dissolved in deionized water)

In the second mutagenicity test if appropriate these top-agar ingredients were preincubated by shaking for approximately 20 min at approximately 30ºC.

After mixing, and pre-incubation if appropriate, the liquid was poured into a petri dish containing a 25 mL layer of minimal agar (i.e., 1.5% (w/v) agar, Vogel-Bonner E medium with 2% (w/v) glucose). After incubation for approximately 48 h at approximately 37°C in the dark, colonies (his+ and trp+ revertants) were counted by hand or by a suitable automatic colony counter.

The counter was calibrated for each test by reading a test pattern plate to verify the manufacturer's requirements for the counter's sensitiveness.

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 induce increases in the mutation frequency which are significant and within the laboratory's normal range.

Criteria for a positive response
A test substance 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 substance 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.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Solubility and toxicity

-The test substance was dissolved in deionized water and a stock solution of 50 mg/mL was prepared for the highest concentration, which provided a final concentration of 5000 µg/plate. Further dilutions of 1,600, 500, 160 and 50 µg/plate were used in the first plate incorporation test.

-The test substance did not precipitate on the plates up to the highest investigated dose of 5,000 µg/plate.

-Because of mutagenic effects with the strain TA 1535 a second plate incorporation test was performed with this strain at with dose levels from 500 to 5,000 µg/mL and the concentrations of 2,000, 3,000 and 4,000 µg/plate were included in the treatment series.

-In both plate incorporation tests toxicity was not observed with and without metabolic activation.

Mutagenicity

-In both independent mutation tests, test substance was tested for mutagenicity with the same stated concentrations. The number of colonies per plate with each strain as well as mean values of 3 plates were given.

-In the absence and in the presence of the metabolic activation system the test substance induced a significant and dose-dependent increase in the number of revertant colonies with the bacterial strain TA 1535.

-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.

Sterility checks and control plates

 

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

Conclusions:

Under the study conditions, the test substance was found to be mutagenic in Salmonella typhimurium TA 1535. All other strains did not show an increase in mutation frequencies.

Executive summary:

An in vitro study was performed to investigate the potential of the test substance to induce gene mutations according to OECD Guideline 471, EPA OPPTS 870.5100 and EU Method B.14, in compliance with GLP.

One independent mutagenicity study was conducted, as the plate incorporation method in strains of Salmonella typhimurium TA 98, TA 100, TA 1535 and TA 1537 and Escherichia coli WP2uvrA. The study was performed in the absence and the presence of a metabolizing system derived from a rat liver homogenate (i.e., S9 mix). The substance was tested for mutagenic effects without and with metabolic activation at five concentrations in the range of 50 - 5000 µg/plate. Additionally the plate incorporation test was repeated with the strain TA 1535 with and without S9-mix at six concentrations in the range of 500 - 5000 µg/plate.

In both plate incorporation tests, the substance induced a dose-dependent increase in the number of revertant colonies with the Salmonella strain TA 1535 in the absence and presence of the S9 mix. Positive controls showed a significant increase in induced mutant colonies, thus indicating the sensitivity of the assay and the efficacy of the S9-mix. No toxicity was observed in any of the assays.

Under the study conditions, the test substance was found to be mutagenic in Salmonella typhimurium TA 1535. All other strains did not show an increase in mutation frequencies.

Genetic toxicity in vivo

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From September 27, 1999 to November 26, 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Gartenstrasse 27, 33178 Borchen
- Age at study initiation: Male/female animals approximately 8 weeks
- Body weight range at treatment: Males: 32-40 g; Females: 27-32 g
- Housing: In fully air-conditioned rooms in makrolon cages type 3 (five animals/cage) on soft wood granulate
- Diet: Rat/mice diet ssnif R/M-H (V 1534), ad libitum
- Water: Tap water in plastic bottles, ad libitum
- Acclimation period: 5 d under study conditions
- Animal identification: Fur marking with KMn04 and cage numbering
- Randomization procedure: Randomization schemes 1999.0432 and 1999.0433

ENVIRONMENTAL CONDITIONS
- Temperature: 22±3°C
- Humidity: 50±20%
- Photoperiod: 12 h light/dark cycle

IN-LIFE DATES: From: To: September 27, 1999 to November 26, 1999

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Deionized water
- Concentration of test material in vehicle: 20% (w/v)
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw

Details on exposure:

PREPARATION OF DOSING SUSPENSION: On the days of administration the test substance was dissolved in deionized water at the appropriate concentration. A magnetic stirrer was used to keep the preparation homogeneous until dosing had been completed.

Duration of treatment / exposure:

2 d

Frequency of treatment:

twice at an interval of 24 h

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5/sex/group

Control animals:

yes, concurrent vehicle

Positive control(s):

Positive control: Endoxan® containing cyclophosphamide
Dissolved in: Distilled water
Dose: 50 mg/kg bw
Concentration: 0.5% (w/v)
Route and frequency of administration: Oral (gavage), once
Volume Administered: 10 mL/kg bw

Tissues and cell types examined:

- 2,000 polychromatic erythrocytes were counted for each animal.  
- The number of cells with micronuclei was recorded, not the number of individual micronuclei.  
- The ratio of polychromatic erythrocytes to 200 total erythrocytes was determined.
- Main parameter for the statistical analysis, i.e. validity assessment of the study and mutagenicity of the test substance, was the proportion of polychromatic erythrocytes with micronuclei out of the 2,000 counted erythrocytes.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Based on the results of preliminary dose range finding study the dose of 2,000 mg/kg bw which did not cause any toxic effects in male and female mice over an observation period of 7 d, was selected as the highest dose, for the main study. This selected dose level also represents the limit dose according to the regulations.

-Preparation and Staining: Animals were killed by carbon dioxide asphyxiation 24 h after dosing. For each animal, about 3 mr fetal bovine serum was poured into a centrifuge tube. Both femora were removed and the bones freed of muscle tissue. The proximal ends of the femora were opened and the bone marrow flushed into the centrifuge tube. A suspension was formed. The mixture was then centrifuged for 5 minutes at approximately 1,200 rpm, after which almost all the supernatant was discarded. One drop of the thoroughly mixed sediment was smeared onto a cleaned slide, identified by project code and animal number and air-dried for about 12 h.

-Staining procedure: The staining was performed as follows:
-5 minutes in methanol
-5 minutes in May-Gronwald's solution
-Brief rinsing twice in distilled water
-10 minutes staining in 1 part Giemsa solution to 6 parts buffer solution, pH 7.2 (Weise)
-Rinsing in distilled water
-Drying
-Coating with Entellan

Evaluation criteria:

Both biological and statistical significances were considered together for evaluation purposes. A test substance is considered positive if there is a significant increase in the number of micronucleated polychromatic erythrocytes compared with the concurrent negative control group. A test substance producing no significant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.

Statistics:

After confirming the validity of the study the following sequential test procedure for the examination of the mutagenicity was applied: Based on a monotone-dose-relationship, one-sided Wilcoxon tests were performed starting with the highest dose group. These tests were performed with a multiple level of significance of 5%.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

orange discolored urine and red discolored feces were observed as clinical signs after administration of test substance

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- All animals survived after treatment. Orange discolored urine and red discolored feces were observed as clinical signs. 24 h after the applications all animals were free of clinical signs.
- The dissection of the animals revealed no test substance related macroscopic findings.
- The bone marrow smears were examined for the occurrence of micronuclei in red blood cells. The incidence of micronucleated polychromatic erythrocytes in the dose group of test substance was within the normal range of the negative control groups. No statistically significant increase of micronucleated polychromatic erythrocytes was observed. The ratio of polychromatic erythrocytes to total erythrocytes remained essentially unaffected by the test substance and was not less than 20% of the control values.
- Cyclophosphamide (i.e., endoxan) induced a marked and statistically significant increase in the number of polychromatic erythrocytes with micronuclei, thus indicating the sensitivity of the test system

Conclusions:

The test substance did not induce micronuclei in bone marrow cells of mice. Therefore, the test substance was not considered to be mutagenic in this micronucleus assay.

Executive summary:

A study was conducted to investigate the potential of the test substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of HsdWin:NMRI mice according to OECD Guideline 474, EPA OPPTS 870.5395 and EU Method B.12, in compliance with GLP.

 

The test substance was dissolved in deionized water and given twice at an interval of 24 h as an oral dose of 2,000 mg/kg bw/day to male and female mice. The dose was selected based on the results of a previous dose range finding study. The animals were sacrificed 24 h after the last administration and bone marrow cells were collected for micronuclei analysis.

The positive control (endoxan) induced a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity of the test system. After treatment with the test substance, the number of polychromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic erythrocytes to total erythrocytes in both male and female animals remained unaffected and was not less than 20% of the control value.

 

Under the study conditions, the test substance did not induce micronuclei in bone marrow cells of mice. Therefore, the test substance was not considered to be mutagenic in this micronucleus assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

An in vitro study was performed to investigate the potential of the test substance to induce gene mutations according to OECD Guideline 471, EPA OPPTS 870.5100 and EU Method B.14, in compliance with GLP. One independent mutagenicity study was conducted, as the plate incorporation method. The study was performed in the absence and the presence of a metabolizing system derived from a rat liver homogenate (i.e., S9 mix). The substance was tested for mutagenic effects without and with metabolic activation at five concentrations in the range of 50 - 5,000 µg/plate. Additionally the plate incorporation test was repeated with the strain TA 1535 with and without S9-mix at six concentrations in the range of 500 - 5,000 µg/plate. In both plate incorporation tests, the substance induced a dose-dependent increase in the number of revertant colonies with theSalmonellastrain TA 1535 in the absence and presence of the S9 mix. Positive controls showed a significant increase in induced mutant colonies, thus indicating the sensitivity of the assay and the efficacy of the S9-mix. No toxicity was observed in any of the assays. Under the study conditions, the test substance was found to be mutagenic in the bacterial reverse mutation assay (Stammberger, 1999).

 

A study was conducted to investigate the potential of the test substance to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster lung according to OECD Guideline 476, EU Method B.17 and EPA OPPTS 870.5300 Method, in compliance with GLP. Two independent experiments were conducted both with and without metabolic activation (i.e., S9 mix). The substance was dissolved in cell culture medium and tested at the concentrations ranging from 50-3,000 µg/mL with and without metabolic activation, based on the results of preliminary testing for solubility and toxicity. Positive controls showed a significant increase in induced mutant colonies, thus indicating the sensitivity of the assay and the efficacy of the S9-mix. The test substance did not induce a relevant reproducible increase in the mutant colonies or mutant frequency three times over the range of the solvent control up to the highest investigated dose in two independent experiments, either with or without metabolic activation by S9-mix. Under the study conditions, the test substance was found to be non-mutagenic in the HPRT assay (Stammberger, 2000a).

 

A study was conducted to investigate the potential of the test substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of HsdWin:NMRI mice according to OECD Guideline 474, EPA OPPTS 870.5395 and EU Method B.12, in compliance with GLP. The test substance was dissolved in deionized water and given twice at an interval of 24 h as an oral dose of 2,000 mg/kg bw/day to male and female mice. The dose was selected based on the results of a previous dose range finding study. The animals were sacrificed 24 h after the last administration and bone marrow cells were collected for micronuclei analysis. The positive control (endoxan) induced a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity of the test system. After treatment with the test substance, the number of polychromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic erythrocytes to total erythrocytes in both male and female animals remained unaffected and was not less than 20% of the control value. Under the study conditions, the test substance did not induce micronuclei in bone marrow cells of mice. Therefore, the test substance was not considered to be mutagenic in this micronucleus assay (Stammberger, 2000b).