2-Naphthalenesulfonic acid, 4-hydroxy-3-[2-[2-methoxy-5-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]diazenyl]-7-[(sulfomethyl)amino]-8-[2-[2-sulfo-4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]diazenyl]-, sodium salt (1:5)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No evidence for genotoxicity was observed in the bacterial reverse mutation assay. The clastogenic evidence observed in the in vitro chromosomal aberration assay at cytotoxic doses were not confirmed or reproduced in the in vivo micronucleus assay in rats.

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

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

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

Test Guideline 111.1

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- pH value in water: approx. 5.5
- Solubility in water: > 100 g/L
- Stability and homogeneity in the solvent: guaranteed for 4 h in deionized water by HPLC analysis
- 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 from rat liver (plate incorporation test) and S9-mix from hamster liver (preincubation test)

Test concentrations with justification for top dose:

Plate incorporation test:
with metabolic activation (10% rat liver):
50, 160, 500, 1600 and 5,000 µg/plate
without metabolic activation:
50, 160, 500, 1600 and 5,000 µg/plate

Preincubation test:
with metabolic activation (30% hamster liver):
16, 50, 160, 500, 1600 and 5,000 µg/plate
without metabolic activation:
16, 50, 160, 500, 1600 and 5,000 µg/plate

Vehicle / solvent:

- Vehicle used: deionized water
- solvent used for positive controls: DMSO

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 (10% rat liver) for all strains

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation (30% syrian golden hamster liver) for strain TA 100, TA 1535, TA 1537 and WP2uvrA

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

congo red

Remarks:

with metabolic activation (30% syrian golden hamster liver) for strain TA 98

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 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 also used. Dose levels used in the second experiment were based on findings, including toxicity, in the first experiment. Toxicity was assessed after microscopic thinning of the bacterial lawn and/or reduction of the number of spontaneously occurring mutants compared to the corresponding solvent control value.

In both tests top agar was prepared which, for the Salmonella strains, contained 100 mL agar (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 (2.5 mL, 2 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 (dissolved in deionized water)

In the second mutagenicity test if appropriate these top-agar ingredients were preincubated by shaking for approximately 20 to 30 minutes at approximately 30°C. After mixing, and preincubation if appropriate, the liquid was poured into a petri dish containing a 25 mL layer of minimal agar (1.5% (w/v) agar, Vogel-BonnerE medium with 2% (w/v) glucose). After incubation for approximately 48 h at approximately 37°C in the dark, colonies (his+ or 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'srequirements for sensitivity.

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, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

(in plate incorporation test and peincubation test)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the plate incorporation test toxicity was observed only with the strain TA 1537 with metabolic activation at concentrations of 500 to 5000 µg/plate and without S9-mix at a concentration of 5000 µg/plate. In the preincubation test no toxicity was seen.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

SOLUBILITY AND TOXICITY: 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 5,000 µg/plate. Further dilutions of 1,600, 500, 160 and 50 µg/plate were used in all experiments. For the preincubation test the concentration of 16 µg/plate was additionally included in the treatment series. Test substance did not precipitate on the plates up to the highest investigated dose of 5,000 µg/plate.
In the plate incorporation test toxicity was observed only with the strain TA 1537 with metabolic activation at concentrations of 500 to 5,000 µg/plate and without S9-mix at a concentration of 5,000 µg/plate. In the preincubation test no toxicity was observed in the absence and in the presence of hamster liver metabolic activation.

MUTAGENICITY:
Plate incorporation test: The test substance did not cause a significant increase in the number of revertant colonies at any dose level with any of the tester strains either in the absence or presence of rat liver S9-mix. No dose-dependent effect was obtained.
Preincubation test: In the presence of hamster liver S9-mix (30% (v/v)) using the preincubation method according to Prival the test substance did not cause a significant or dose dependent increase in the number of revertant colonies under the experimental conditions described.

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. The number of revertant colonies of the positive control in the preincubation test with the strain TA 1537 in the presence ofS9-mix was even slightly above ofthe historical control data range, but the criteria for the positive response were clearly fulfilled and the validity not influenced.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

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 (background control and positive controls) gave the expected number of colonies, i.e. values were within the laboratory's historical control range. The number of revertant colonies of the solvent and negative controls with the strain WP2uvrA in the presence of S9-mix in both experiments was marginally below the historical control data range, which had no influence on the validity of the assay.

Conclusions:

Interpretation of results:
negative with metabolic activation
negative without metabolic activation

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

Executive summary:

The test substance was investigated for its potential to induce gene mutations according to OECD Guideline 471, EPA OPPTS 870.5100, EU Method B.14 and Japan: Guidelines for Screening Mutagenicity Testing of Chemicals, Test Guidelines 111.1, in compliance with GLP.

Two independent mutagenicity studies were conducted, one with the plate incorporation method and the other as a modified preincubation test (Prival test). The studies were performed in the absence and in the presence of a metabolizing system derived from rat or hamster liver homogenate. The substance was assessed at 50 - 5,000 µg/plate in the standard plate test and 16 - 5,000 µg/plate in the modified preincubation test.

The substance did not cause a significant increase in the number of revertant colonies at any dose level in the absence or presence of metabolic activation system in either of the tests.

Under the study conditions, the substance was found to be non-mutagenic in the bacterial reverse mutation assay.

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:

From Apr. 04, 2005 to May 31, 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - 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

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

Test Guideline 111.2

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

- Stability and homogeneity in the solvent: not necessary because the test substance was dissolved directly into the test system (MEM (minimal essential medium))
- Concentration of stock solution: 5 mg/mL
- Solubility: Solution in cell culture medium

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from rat liver

Test concentrations with justification for top dose:

First experiment with 3/20 h treatment/sampling time:
without S9-mix: 39.1, 78.2, 156.3, 312.5, 625$, 1250$, 2500$ and 5000$ µg/mL
with S9-mix: 39.1, 78.2, 156.3, 312.5, 625, 1250$, 2500$ and 5000$ µg/mL
$ = concentrations at which metaphase analysis was conducted

Vehicle / solvent:

cell culture medium

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

(without metabolic activation) at the dose of 1500 µg/mL

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

(with metabolic activation) at the dose of 7.5 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 h
- Sampling time: 20 h

CELL CULTURE:
Large stocks of the mycoplasma-free V79 cell line were stored in liquid nitrogen which permitted repeated use of the same cell culture batch for numerous experiments. The identical characteristics of the cells ensured 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 100000 to 1 X 1000000 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. The cells were subcultured twice a week.

ASSAY PROCEDURE:
Toxicity experiments and dose range finding:
For the determination of cytotoxic effects cell cultures on slides were treated with the test substance. Evaluation of cell number was performed in the first experiment with the 3/20 h treatment/sampling time with and without S9-mix. In the second experiment cell evaluation was performed with the 20/20 h and 28/28 h treatment/sampling time without S9-mix and with 3/28 h treatment/sampling time with S9-mix. Using a 500 fold microscopic magnification the cells were counted in 10 fields of the slides. The cell number of the treatment groups was presented as % cells in relation to the control. The test included the following treatments:
Solvent control : the maximum final concentration of organic solvents was approx. 1% (v/v).
Test substance: the highest dose level was determined by the solubility of the test substance up to the maximum of 10 mM, or the international limit dose, 5000 µg/mL. Treatments were performed both in the presence and absence of the S9-mix metabolic activation system using a duplicate cell culture at each test point.

Rationale for dose selection:
The evaluated concentrations for mutagenicity were based on the results of the cell counting. For non-toxic, freely soluble test substances, the top dose is either 10 mM or 5,000 µg/mL according to international testing guidelines. For relatively insoluble test substances that are not toxic at concentrations lower than the insoluble concentration, the highest dose used is a concentration above the limit of solubility in the final culture medium after the end of the treatment period. For toxic substances, a highest dose level is selected which reduced survival and/or the mitotic index below 50%, of the corresponding solvent control.

Mutagenicity test:
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 recorded. Exponentially growing cultures which were more than 50% confluent were trypsinated by an approx. 0.25% (v/v) trypsin solution ready for use.

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 Quadriperm® dishes which were then seeded with cells to yield 3-4 x 10000 cells/slide. Thus for each dose level and treatment time, duplicate cultures slides were used. The Quadriperm® dishes contained 6 mL MEM with approx. 10% (v/v) FCS and approx. 0.1% (w/v) neomycinsulfate.
After 48 h, the medium was replaced with one containing approx. 10% (v/v) FCS and the test substance, or positive control, or solvent and in the presence of metabolic activation additionally 2% ( v/v) S9-mix.

For the 3 h treatment time, the medium was replaced by normal medium following two rinses. In the second experiment the cells were exposed to the treatment medium without S9-mix for 20 h and 28 h. 18 h and 26 h after the start of the treatment, Colcemide was added (approx. 0.05 µg/mL/culture medium) to the cultures to arrest mitosis and 2 h later (20 h and 28 h after the start of treatment) metaphase spreads were prepared as follows:
The cultures were made hypotonic by adding about 5 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 2 mL fixative.

Then the liquid was replaced by 6 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 air dried for 24 h. 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 Entellan® or Corbit®

Evaluation criteria:

Analysis of metaphases:
The slides were coded and 25-100 metaphases/group and cell culture were examined. The set of chromosomes was examined for completeness and the chromosomal aberrations were assessed and classified. Only metaphases with 22+/-2 chromosomes were included in the analysis and were examined for the following aberrations: chromatid gap, chromosome gap, chromatid break, chromosome break, acentric chromatids (chromosomes), chromatid deletion, chromosome deletion, chromatid exchanges, chromosome exchanges including intrachanges, dicentrics and ring formation, pulverization and multiple aberrations.
Furthermore the incidence of polyploid metaphases and endoreduplications was determined in 500 metaphase cells of each cell culture. The rate of the polyploid cells and endoreduplications was expressed as a percentage. Additionally the mitotic index (expressed as %) was determined by counting the number of cells undergoing mitosis in a total of 1,000 cells. The results from the dose groups were compared with those of the control group and the positive control at each sampling time.

Criteria for a valid assay:
The assay is considered valid if the solvent control data are within the laboratory's normal control range for the spontaneous mutant frequency and positive control induce increases in the mutation frequency which are statistically significant and within the laboratory's normal range.

Criteria for clastogenicity:
The test substance is classified as nonclastogenic if the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of historical control data and/or no significant increase in the number of structural chromosome aberrations is observed.
The test substance is classified as clastogenic if number of induced structural chromosome aberrations is above the range of historical control data and either a concentration-related or a significant increase in the number of structural chromosome aberrations is seen.

Statistics:

The Biometry of the results was performed with a one-sided Fisher's exact test.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

After treatment with the test substance there was no relevant increase in the number of polyploid cells as compared with the solvent controls, but there was a distinct enhancement of endoreduplicated cells in the highest evaluated and severely cytotoxic concentration.

There was a dose-dependent enhancement of the aberration rates at the 3/20 h treatment/sampling time with and without S9-mix. These data were found significantly enhanced in the Fisher's exact-test.

In the absence of S9-mix clastogenicity in form of aberration rates above the historical solvent control data range was only observed in the cytotoxic concentration range. The concentration 2,500 µg/mL (- S9-mix) showed reduced cell survival (46.5%) and caused a slight increase in aberrations. The severely toxic concentration group 5,000 µg/mL was additionally evaluated confirming that the effect is toxicity and concentration-related.

In the presence of S9-mix, with the lowest evaluated (non-toxic) concentration 1,250 µg/mL the phases with aberrations exclusive gaps were only marginally above the historical control data range and therefore rather equivocal, but the increase was statistically significant. Higher concentrations causing clear cytotoxicity produced a more distinct increase in aberration rates.

On the basis of these findings a second experiment was not required.

The sensitivity of the test system was demonstrated by the enhanced mutation frequency in the cell cultures treated with the positive control items.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

SOLUBILITY AND TOXICITY TESTING:

The test substance was dissolved in MEM. Evaluation of the solubility of that solution in MEM Earle's salts cell culture medium showed that 5000 µg/mL was a practicable concentration and produced no precipitate. Evaluation of cytotoxicity by cell counting showed that the test substance was severely toxic to the V79 cells in the absence and in the presence of metabolic activation (S9-mix) after 3 h treatment time. At the highest dose level, 5000 µg/mL, without S9-mix survival was reduced to 32.4% of the solvent control value. In the presence of metabolic activation (S9-mix) at the concentration of 5000 µg/mL survival was reduced to 29.7% of the solvent control value. Evaluated concentration groups showed no relevant reduction of the mitotic index except of the highest concentration group without S9-mix, where the mitotic index was mildly reduced after treatment (72.3% of the control). Before treatment, the pH values and osmolality of the treatment media were determined. The addition of test substance solutions did not have any relevant effect on these parameters.

Conclusions:

Interpretation of results:
positive with and without S9-mix in correlation with cytotoxicity

Under the test conditions, the test substance induced structural chromosome aberrations in V79 Chinese hamster cells at cytotoxic concentrations in the presence and absence of a metabolic activation system.

Executive summary:

A study was conducted to investigate the potential of the test substance to induce chromosome aberrations in V79 cells of the Chinese hamster lung fibroblasts according to OECD Guideline 473, EPA OPPTS 870.5375, EU Method B.10 and Japanese Guideline 111.2, in compliance with GLP.

 

The test substance was dissolved in cell culture medium and tested at the concentrations ranging from 39.1-5,000 µg/mL with and without metabolic activation, based on the results of preliminary testing for solubility and toxicity.

 

The test substance was found to be severely cytotoxicto the V79 cells in the presence and absence of metabolic activation (S9-mix) after 3 h treatment time. At the highest dose level (5,000 µg/mL), the cell survival was reduced to 29.7% and 32.4% of the solvent control value in the presence and absence of metabolic activation system respectively.

 

The test substance induced a significant and dose-dependent increase in structural chromosome aberration rates at the 3 h treatment time with and without S9-mix in correlation with cytotoxicity. The high cytotoxic concentration (5,000 µg/mL) induced an enhancement of endoreduplicated cells. Positive controls showed a significant increase in chromosome aberrations, thus indicating the sensitivity of the assay and the efficacy of the S9-mix.

 

Under the test conditions, the test substance induced structural chromosome aberrations in V79 Chinese hamster cells at cytotoxic concentrations in the presence and absence of a metabolic activation system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The test substance was not clastogenic in the micronucleus assay in mice

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From June 07, 2005 to June 09, 2005

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:

other: ICH Guidelines S2A and S2B

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

other: CRL: NMRI BR mice

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (Europe) Laboratories, Budapest, HUNGARY
- Age at study initiation: young adult mice
- Weight at study initiation: male: 26.8-32.1 g and female: 22.5-25.8 g
- Assigned to test groups randomly: yes
- Housing: group caging (5 animals/cage), type II polypropylene/polycarbonate cages with laboratory bedding
- Diet: SSNIFF RIM-Z+H diet for rats and mice (ad libitum)
- Water: ad libitum
- Acclimation period: 6 d

ENVIRONMENTAL CONDITIONS
- Temperature: 22±3°C
- Humidity: 30-70 %
- Photoperiod: 12 h dark/light

IN-LIFE DATES: From: June 07, 2005 To: June 09, 2005

Route of administration:

oral: gavage

Vehicle:

Vehicle used for test substance: distilled water
- Concentration of test substance in vehicle: 20% (w/v)
- Batch no.: 9240604
- Date of expiry: June, 2007
- Storage condition: at room temperature

Vehicle of positive control: 0.9% NaCl infusion
- Batch no.: 5671002
- Expiry: October, 2005
- Storage condition: at room temperature

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test substance was dissolved in distilled water for the treatment. The necessary amount of the test substance was weighed into a calibrated volumetric flask and distilled water was added and stirred to obtain a homogenous formulation. It was diluted to the final volume with the distilled water. The solution was prepared fresh each day of dosing and used within 2 h. The test substance was used for treatment at concentration of 20% (w/v). Cyclophosphamide (positive control) was dissolved in 0.9% NaCI infusion for treatment.

Duration of treatment / exposure:

48 h

Frequency of treatment:

twice

Post exposure period:

24 h after the last dosing

Remarks:

Doses / Concentrations:
2000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

5/sex

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: intraperitoneally
- Doses: 60 mg/kg
- Animals used: 5/sex

Tissues and cell types examined:

The slides were examined in a blind manner. Two thousand polychromatic erythrocytes were scored per animal to asses the micronucleatcd cells. The frequency of micronucleated cells were expressed as percent of micronucleated cells based on the first 2000 polychromatic erythrocytes counted in
the optic field. Multiple micronuclei cells were not registered. The proportion of immature among total (irnmature+mature) erythrocytes were
determined for each animal by counting a total of at least 200 immature erythrocytes.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Based on the results of the acute oral toxicity study the dose of 2,000 mg/kg bw was selected as the limit dose, for the main study.

TREATMENT AND SAMPLING TIMES: The test substance was administered twice at an interval of 24 h orally by gavage to the test animals at a dose 2,000 mg/kg bw. The treatment volume was 0.1 mL/kg bw. The vehicle, distilled water was administered in the same way to the negative control groups. The mice were examined for visible signs of reactions to treatment, immediately after dosing, and at intervals until sacrifice. Sampling was made once at 24 h after the last dosing. Five male and female animals per group were used for sampling.
Cyclophosphamide (positive control) was administered intraperitoneally with a treatment volume: 0.1 mL/kg bw. Sampling was performed 24 h after the beginning of the treatment and five male and female animals were used for sampling.

DETAILS OF SLIDE PREPARATION: Bone marrow was obtained from two exposed femurs of the mice from animals immediately after sacrificing. The bone marrow was flushed with foetal calf serum. The cells were concentrated by a gentle centrifugation, then spread on a standard microscopic slide. Slides were fixed and after that stained with Giemsa. Two slides were prepared from each animal.

EVALUATION OF THE RESULTS: The micronucleated polychromatic erythrocytes referring to 2,000 polychromatic erythrocytes and the ratio of polychromatic erythrocytes to normochromatic erythrocytes (PCE/NCE) were listed for each animal. The frequency of micronucleated cells of treated male and female groups were compared with those of the vehicle control groups.

Evaluation criteria:

A positive result was one that gave a significant increase (at p<0.05, p<0.01) of the frequency of micronucleated polychromatic erythrocytes at least in two treated groups in one sex.
This increase would be classified as significant if observed:
- at adjacent dose levels: in the same experiment, dose- dependency,
- at adjacent time points: in the same experiment, same dose level, that is time dependency,
- in two experiments, in the same dose level and time point, that is reproducibility.
Both biological and statistical significance were considered together for evaluation purposes.
The historical range for this laboratory was also considered when evaluating the biological significance of small increases.

Statistics:

Dose dependent increase in the number of micronucleated polychromatic erythrocytes was evaluated by Kruskal-Wallis Non Parametric ANOVA test at 1 and 5% probability levels.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Clinical signs and mortality:

No animals died during the study. Animals did not show any toxicity symptom in the test substance treated group or in the vehicle or positive control groups. Due to the effect of test substance the faeces and tissue of abdominal organs of mice were found to be discoloured with a reddish appearance. The presence of red colour in the tissue after an oral exposure demonstrates systematic exposure to the test substance.

Frequency of micronucleated polychromatic erythrocytes:

The frequency of micronucleated polychromatic erythrocytes for the vehicle control mice was within an acceptable range and compatible with the historical control data. Cyclophosphamide treated mice (60 mg/kg bw) showed large, statistically significant increase in the micronucleated polychromatic erythrocytes number compared to the vehicle control, demonstrating an acceptable sensitivity of the test. The repeated administration of the test substance (twice at an interval of 24 h) at 2,000 mg/kg bw did not induce biologically relevant increase in the frequency of micronucleated polychromatic erythrocytes (MPCEs) in male or female mice at 24 h after the treatment compared to the vehicle control. In the female animals, at 24 h after treatment with 2,000 mg/kg test substance there was small, statistically significant, but biologically not important increase in the number of micronucleated polychromatic erythrocytes (MPCEs). These minor increases were in the range of historical negative control value. No significant difference was found in the ratio of polychromatic and normochromatic erythrocytes after treatment.

Conclusions:

Interpretation of results: negative
Under the test conditions, the test substance did not induce micronuclei in bone marrow cells of the treated mice. Therefore, the test substance was not considered to be clastogenic 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 mice according to OECD Guideline 474 and ICH Guidelines S2A and S2B, in compliance with GLP.

The substance was dissolved in distilled water and given twice at an interval of 24 h as an oral dose of 2,000 mg/kg bw to male and female rats. The dose was selected based on the results of a preliminary acute oral toxicity study. A vehicle control group treated with distilled water and a positive control group treated with cyclophosphamide (60 mg/kg bw intraperitoneally) were also included. The animals were sacrificed 24 h after the last administration and bone marrow cells were collected for micronuclei analysis. Two thousand polychromatic erythrocytes (PCEs) were scored per animal to assess the micronucleated cells. The ratio of polychromatic to normochromatic erythrocytes (PCE/NCE) was used to assess the toxicity of the test substance.

The oral administration did not induce any biologically relevant increase in the frequency of micronucleated polychromatic erythrocytes (MPCEs) in male or female mice compared to the vehicle control. No biologically important alteration in the ratio of polychromatic to normochromatic erythrocytes occurred in the treated groups when compared to the vehicle control. Treatment with the positive control, cyclophosphamide, caused significant increase in the number of micronucleated polychromatic erythrocytes (MPCEs), thus validating the test.

Under the study conditions, the test substance did not induce micronuclei in bone marrow cells of the treated mice. Therefore, the test substance was not considered to be clastogenic in this micronucleus assay (Beres, 2005).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro:

The test substance was investigated for its potential to induce gene mutations according to OECD Guideline 471, EPA OPPTS 870.5100, EU Method B.14 and Japanese Guidelines 111.1, in compliance with GLP. Two independent mutagenicity studies were conducted, one with the plate incorporation method and the other as a modified preincubation test (Prival test). The studies were performed in the absence and in the presence of a metabolizing system derived from rat or hamster liver homogenate. The substance was assessed at 50 - 5,000 µg/plate in the standard plate test and 16 - 5,000 µg/plate in the modified preincubation test. The substance did not cause a significant increase in the number of revertant colonies at any dose level in the absence or presence of metabolic activation system in either of the tests. Under the study conditions, the substance was found to be non-mutagenic in the bacterial reverse mutation assay (Kaufmann, 2005g).

A study was conducted to investigate the potential of the test substance to induce chromosome aberrations in V79 cells of the Chinese hamster lung fibroblasts according to OECD Guideline 473, EPA OPPTS 870.5375, EU Method B.10 and Japanese Guideline 111.2, in compliance with GLP. The test substance was dissolved in cell culture medium and tested at the concentrations ranging from 39.1-5,000 µg/mL with and without metabolic activation, based on the results of preliminary testing for solubility and toxicity. The substance was found to be severely cytotoxic to the V79 cells in the presence and absence of metabolic activation (S9-mix) after 3 h treatment time. At the highest dose level (5,000 µg/mL), the cell survival was reduced to 29.7% and 32.4% of the solvent control value in the presence and absence of metabolic activation system, respectively. The test substance induced a significant and dose-dependent increase in structural chromosome aberration rates at the 3 h treatment time with and without S9-mix in correlation with cytotoxicity. The high cytotoxic concentration (5,000 µg/mL) induced an enhancement of endoreduplicated cells. Positive controls showed a significant increase in chromosome aberrations, thus indicating the sensitivity of the assay and the efficacy of the S9-mix. Under the test conditions, the test substance induced structural chromosome aberrations in V79 Chinese hamster cells at cytotoxic concentrations in the presence and absence of a metabolic activation system (Kaufmann, 2005h).

In vivo:

A study was conducted to investigate the potential of the test substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of mice according to OECD Guideline 474 and ICH Guidelines S2A and S2B, in compliance with GLP. The substance was dissolved in distilled water and given twice at an interval of 24 h as an oral dose of 2,000 mg/kg bw to male and female rats. The dose was selected based on the results of a preliminary acute oral toxicity study. A vehicle control group treated with distilled water and a positive control group treated with cyclophosphamide (60 mg/kg bw intraperitoneally) were also included. The animals were sacrificed 24 h after the last administration and bone marrow cells were collected for micronuclei analysis. Two thousand polychromatic erythrocytes (PCEs) were scored per animal to assess the micronucleated cells. The ratio of polychromatic to normochromatic erythrocytes (PCE/NCE) was used to assess the toxicity of the test substance. The oral administration did not induce any biologically relevant increase in the frequency of micronucleated polychromatic erythrocytes (MPCEs) in male or female mice compared to the vehicle control. No biologically important alteration in the ratio of polychromatic to normochromatic erythrocytes occurred in the treated groups when compared to the vehicle control. Treatment with the positive control, cyclophosphamide, caused significant increase in the number of micronucleated polychromatic erythrocytes (MPCEs), thus validating the test. Under the study conditions, the test substance did not induce micronuclei in bone marrow cells of the treated mice. Therefore, the test substance was not considered to be clastogenic in this micronucleus assay (Beres, 2005).

Justification for classification or non-classification

The test substance was evaluated for its genotoxic potential in a bacterial reverse mutation assay, anin vitrochromosomal aberration assay and anin vivomicronucleus assay. No evidence for genotoxicity was observed in the bacterial reverse mutation assay. In thein vitrochromosomal aberration test, although there was evidence for clastogenicity, this was observed at doses that were also moderately cytotoxic. Further, no genotoxicity was observed in thein vivomicronucleus assay in rats. Therefore, as the evidence of clastogenicity was only observed at cytotoxic concentrations in thein vitrostudy and not in thein vivoassay, the test substance does not require classification for mutagenicity according to EU CLP criteria (EC 1272/2008).