Disodium 6-(4,6-dichloro-1,3,5-triazin-2-ylamino)-1-hydroxy-2-(4-(2-(sulfonatooxy)ethylsulfonyl)phenylazo)naphthalene-3-sulfonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance did not show mutagenic effects in an Ames Prival assay; however showed cytogenic effects at cytotoxic concentrations in V79 cells in an in-vitro chromosome aberration assay. This positive effects was considered to represent a false positive effect, as the in-vivo micronucleus test is clearly negative.

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:

1999-02-17-1999-03-26

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 (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Test organism:
Salmonella typhinium strains:
TA98 hisD3052 rfa uvrB + R
TA 100 hisG46 rfa uvrB +R
TA 1535 hisG46 rfa uvrB,
TA 1537 hisC3076 rfa uvrB
and
E. coli WP2 uvrA pkm101

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):

Bacteria:
The strains of Salmonella typhimurium were obtained from professor B.N. Ames, University of California, U.S.A. The strain of E. coli was obtained from the national Collection of Industrial Bacteria, Aberdeen, Scotland.
Bacteria were grown overnight in nutrient broth (25 g Oxid 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 requirements, membrane permeability, ampicillin resistance, crystal violet sensitivity, UV resistance and response to diagnostic mutagens. All criteria for a valid assay were fulfilled.

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix from induces rat liver and uninduced hamster liver

Test concentrations with justification for top dose:

Concentration range (all tests): 50, 160, 500, 1600, and 5000 µg/plate

Vehicle / solvent:

Solvent: distilled water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

congo red

other: 2-aminoanthracene, 1-methyl-3-nitro-1-nitrosoguanidine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation; in suspension;

Test System:
Test Groups: PLATE INCORPORATION TEST
a) Without metabolic activation: 50; 160, 500, 1600 and 5000 µg/plate
b) With metabolic activation (10 % rat liver): 50, 160, 500, 1600 and 5000 µg/plate
PREINCUBATION TEST
a) Without metabolic activation: 50; 160, 500, 1600 and 5000 µg/plate
b) With metabolic activation (30 % rat liver): 50, 160, 500, 1600 and 5000 µg/plate


CONTROL GROUPS:
Negative controls:
a) Untreated control
b: solvent control
Positive controls:
a) without metabolic activation (Sodium-azide for Strain TA 100 and TA 1535, 9 aminoacridine for strain TA 1537, 2-Nitrofluorene for strain 98, MNNG for strain WP2uvrA and 4-NQO for strain WP2uvrA
b): with metabolic activation (10 % rat liver)
2-aminoanthracene for all tester strains
c)-with metabolic activation (30 % Syrian golden hamster liver and preincubation)
2-aminoanthracene for strain TA 100, TA 1535 and TA 1537
Congo red for Strain TA98

Formulation of test compound: dissolved in deionized water at appropriate concentrations immediately before use.

DURATION
- Preincubation period: 30 min preincubation in the presence of 30 % (v/v) Syrian golden hamster S9-mix.
Three volumes of S9 fraction was mixed with 7 volumes of the S9 cofactor solution.

Evaluation criteria:

Assay considered valid if:
-Solvent control data are within the laboratory´s normal control range for the spontaneous mutant frequency
-Positive controls induced increases in the mutation frequency which were both statistically significant and within the laboratory´s normal range
Test compound is classified as mutagenic if has either following effects:
-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
-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 test substance does not achieve either of the above criteria, it is considered to show no evidence of mutagenic activity in this system Sterility of S9-mix and the test compound were indicated by the absence of contamination on the test material 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 laboratories control range. Test compound proved to be not toxic to the bacterial strains. In all independent mutation tests, the test substance was tested for mutagenicity with concentrations as described in table above. Number of colonies per plate with each strain as well as mean values of 3 plates is given.

Species / strain:

S. typhimurium TA 1535

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 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 pKM 101

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: NA

- Water solubility:
The test compound did not precipitate on the plates until the highest investigated dose of 5000 µg/plate
The test substance proved to be not toxic to the bacterial strains.
HISTORICAL CONTROL DATA (see table under "overall remarks”)

STERILITY CHECKS AND CONTROL PLATES

Sterility of S9-mix and the test material and S9-mix sterility check plates. Control plates gave the expected number of colonies, i.e. values were within the laboratory's historical range.

SOLUBILITY AND TOXICITY

The test compound 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 1600, 500, 160 and 50 µg/plate were used in all experiments.

The test compound proves to be not toxic to the bacterial strains.

MUTAGENICITY:

In all independent mutation tests, the test substance was tested for mutagenicity with the same concentration.

 

AMES 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 in the presence of rat liver S9-mix in either mutation test. No dose dependant effect was obtained.

PRIVAL 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 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.

Conclusions:

The test substance is not mutagenic in the standard plate test; Ames Test as well as in the preincubation method according to Prival.

Executive summary:

The substance was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537 and TA 98 of Salmonella typhimurium and Escherichia coli WP2uvrA.

Two independent mutagenicity studies were conducted one standard plate test ( Ames Test) and a modified preincubation test (Prival Test). The studies were performed in the absence and in the presence of a metabolizing system derived from a rat liver homogenate or a hamster liver homogenate. Additionally, a repeat of the preincubation test was performed with the strain WP2uvrA in the absence of S9 -mix.

For all studies, the compound was dissolved in deionized water, and each bacterial strain was exposed to 5 dose levels. Doses for all studies ranged from 50 to 5000 µg/plate.

Control plates without mutagen showed that the number of spontaneous revertant colonies was within the laboratory's historical control range and similar to that described in the literature. All the positive control compounds gave the expected increase in the number of revertant colonies, except in the first preincubation test with the strain WP2uvrA in the absence of S9-mix, where the positive control showed not the expected increase in the number of revertant colonies. This questionable effect was caused by a decomposition of N-Methyl-N-nitrosoguanidine (MNNG). In a repeat of this strain with an alternative positive compound 4 -Nitroquinoline-N-Oxide (4 -NQO) the sensitivity of the assay could be demonstrated.

Toxicity: In the plate incorporation test and also in the preincubation test toxicity was not observed either with or without metabolic activation.

Ames Test:

Mutagenicity: in the absence of the metabolic activation system the test compound did not result in relevant increases in the number of revertants in any of the bacterial strains. Also in the presence of rat liver activation system (10 % (v/v)), treatment of the cells with the test substance did not result in relevant increases in the number of revertant colonies.

 

Prival Test:

In the presence of hamster liver S9-mix (30 % (v/v)) using the preincubation method according to Prival the test compound did not cause a significant increases in the number of revertant colonies with any of the tester strains under experimental conditions described.

Therefore, the test substance is not mutagenic in the standard plate test (Ames Test) as well as in the preincubation method according to Prival at the investigated dose levels.

 

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 Oct. 06, 1997 to Jan. 09, 1998

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

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Source: cell bank of "Genetic Toxicology", Hoechst AG
Cell culture medium: MEM (minimal essential medium) with Hankssalts and 25 mM Hepes-buffer
pH values and osmolality of the treatment media: Determined before treatment

Cytokinesis block (if used):

Colcemide

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Experiment with 20 h preparation time:
without S9-mix: 10#, 25#, 50, 100, 250# and 500 µg/mL
with S9-mix: 100#, 250, 500, 1000 and 1500 µg/mL

Experiment with 28 h preparation time:
without S9-mix: 50#, 100#, 250# and 500 µg/mL
with S9-mix: 500#, 1000 and 1500* µg/mL

# not used because higher concentrations were evaluated
* not evaluated because of high toxicity

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

(without metabolic activation )

Positive control substance:

ethylmethanesulphonate

Remarks:

(5000 µg/mL- 20 h treatment)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

(with metabolic activation )

Positive control substance:

cyclophosphamide

Remarks:

(3.5 µg/mL)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3h (Experiment 1, with and without S9-mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 20 h

SPINDLE INHIBITOR (cytogenetic assays): Colcemide (approx. 0.05 µg/mL /culture medium) The cells were prepared 2 h later (20 and 28 h preparation)
STAIN (for cytogenetic assays): 2 % (w/v) Orcein solution

NUMBER OF REPLICATIONS: Two

NUMBER OF CELLS EVALUATED: 25 - 100 metaphases per experimental group and cell culture; 1000 cells of each cell culture

DETERMINATION OF CYTOTOXICITY
- Method: Mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes

Evaluation criteria:

The test substance is classified as clastogenic
(a) If it induces a reproducible statistically significant increase in the number of phases with aberrations (without gaps) with one or more of the concentrations tested as compared with the solvent controls.
(b) If there is a reproducible concentration-related increase in the number of phases with aberrations (Without gaps).

The test substance is classified as non-clastogenic if the tests are negative both with and without metabolic activation.

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:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No effects
- Effects of osmolality: No effects

RANGE-FINDING/SCREENING STUDIES:
Evaluation of the solubility of the suspension in cell culture medium showed that 5000 µg/mL was the highest dose level tolerated for the test system.

Preliminary toxicity study: Was carried out using a maximum concentration of 5000 µg/mL and a range of lower dose levels down to 10 µg/mL.

Following treatment in the absence and in the presence of S9 metabolic activation, high toxicity was observed at 2500 µg/mL and above. Survival declined in a dose-related manner reaching 18.2 % of the solvent control value at the highest dose level, 5000 µg/mL, in the absence of S9-mix and 9.9 % with S9-mix

COMPARISON WITH HISTORICAL CONTROL DATA: Yes

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 substances.

 

Mutation results:

In the absence of metabolic activation the aberration rate was enhanced in the first experiment 20 h after treatment, but only inclusive gaps.

In the second experiment the number of phases with aberrations was increased inclusive and exclusive gaps. Also at the 28 h preparation time in both experiments an enhanced aberration rate with and without gaps was observed (5000 µg/mL).

In the presence of S9-mix an increased number of cells with aberrations inclusive gaps was found at the 20 h preparation time in the second experiment at a concentration of 1500 µg /mL, while at the 28 h preparation time an increased number of phases with aberrations inclusive and exclusive gaps were found at the highest evaluable dose level of 1000 µg /mL in both experiments

 

These data were found significantly enhanced in the Fisher's exact-test. In addition, an increased number of exchange figures was observed.

This is an indication of chromosomal damage. The sensitivity of the test system was demonstrated by the enhanced mutation frequency in the cell cultures treated with the positive control compounds.

Conclusions:

Under the test conditions, the test substance was clastogenic in the in vitro chromosome aberration assay with V79 Chinese hamster lung cells.

Executive summary:

A study was conducted to investigate the potential of test substance to induce chromosome aberrations in V 79 cells of the Chinese hamster lung in vitro according to OECD Guideline 473, EPA OPPTS 870.5375 and EU Method B.10. in compliance with GLP.

The test substance was dissolved in DMSO and tested at the following concentrations:

First experiment with 20 h treatment time:

without S9-mix: 10, 25, 50, 100, 250 and 500 µg/mL

with S9-mix: 100, 250, 500, 1000 and 1500 µg/mL

Second experiment with 28 h treatment time:

without S9-mix: 10, 25, 50, 100, 250 and 500 µg/mL

with S9-mix: 100, 250, 500, 1000 and 1500 µg/mL

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.

In the highest investigated dose the test compound induced a significant increase in the number of chromosome aberrations in the presence and in the absence of metabolic activation.

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.

Test substancedoes induce chromosome aberrations in V79 Chinese hamster cells, both in the presence as well as in the

absence of a metabolic activation system.

Under the test conditions, the test substance was clastogenic in the in vitro chromosome aberration assay with V79 Chinese hamster lung cells.

Reference 3

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 Aug. 27, 1997 to Oct. 24, 1997

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 OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Plate incorporation test:
a: without metabolic activation:
4, 20, 100, 500, 2500, 5000 µg/plate
b: with metabolic activation:
4, 20, 100, 500, 2500, 5000 µg/plate

Preincubation test:
a: with metabolic activation (30 % Syrian golden hamster liver and preincubation):
4, 20, 100, 500, 2500, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Water
- Justification for choice of solvent/vehicle: Soluble in water

Untreated negative controls:

yes

Remarks:

(Untreated controls)

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

(without metabolic activation)

Positive control substance:

sodium azide

Remarks:

(for strain TA 100 and TA 1535)

Untreated negative controls:

yes

Remarks:

(Untreated controls)

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

(without metabolic activation)

Positive control substance:

9-aminoacridine

Remarks:

(for strain TA 1537)

Untreated negative controls:

yes

Remarks:

(Untreated controls)

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

(without metabolic activation)

Positive control substance:

2-nitrofluorene

Remarks:

(for strain TA 98)

Untreated negative controls:

yes

Remarks:

(Untreated controls)

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

(with metabolic activation-10 % rat liver)

Positive control substance:

other: 2-aminoanthracene for all the strains

Remarks:

None

Untreated negative controls:

yes

Remarks:

(Untreated controls)

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

(with metabolic activation-30 % Syrian golden hamster liver and preincubation)

Positive control substance:

other: 2-aminoanthracene for strain TA 100, TA 1535, TA 1537

Remarks:

None

Untreated negative controls:

yes

Remarks:

(Untreated controls)

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

(with metabolic activation-30 % Syrian golden hamster liver and preincubation)

Positive control substance:

congo red

Remarks:

(for strain TA 98)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation

DURATION
- Incubation period: 48 h at approx. 37 °C


NUMBER OF REPLICATIONS: Three

DETERMINATION OF CYTOTOXICITY
- Method: 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.

Evaluation criteria:

The test substance was considered positive if
(a) 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) 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:

Not reported (According to the OECD guideline 471, a statistical analysis of the data was not mandatory).

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not examined

Untreated negative controls validity:

not examined

Positive controls validity:

not examined

Species / strain:

S. typhimurium TA 1535

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 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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test substance did not precipitate on the plates up to the highest investigated dose of 5000 µg/plate.


COMPARISON WITH HISTORICAL CONTROL DATA: Yes

Sterility checks and control plates:

Sterility of S9-mix and the test substance were indicated by the absence of contamination on the test material 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.

Solubility and toxicity:

The substance did not precipitate on the plates up to the highest investigated dose of 5000 µg/plate. The test substance proved to be toxic to the bacterial strain TA 1537 at concentration of 2500 µg /plate in the absence of metabolic activation and at a concentration of 5000 µg /plate in the presence of rat metabolic activation, but only in the first mutation experiment.

A toxicity test using histidine-enriched agar plates and a dilution of the tester strain TA 100 was performed in parallel with the second experiment. The test compound proved to be not toxic to the bacterial strain.

Mutagenicity

Plate incorporation test:

In the plate incorporation test the test compound did not cause a significant increase in the number of revertant colonies with any of the tester strains either in the absence or in the presence of 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 increase in the number of revertant colonies.

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.

Conclusions:

Under the test conditions, the test substance is not mutagenic both in the presence and absence metabolic activation in the standard plate test (Ames Test) and in the preincubation method according to Prival.

Executive summary:

A study was conducted to determine the mutagenic potential of test substance according to OECD Guideline 471, EPA OPPTS 798.5265 and EU method B.13/14. in compliance with GLP.

Strains TA 100, TA 1535, TA 1537 and TA 98 of Salmonella typhimurium were used in the mutagenicity assay.

Two independent mutagenicity studies were conducted, one as the standard plate test 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 a rat liver homogenate or a hamster liver homogenate. For both studies, the compound was dissolved in aqua bidest., and each bacterial strain was exposed to 6 dose levels.

Doses for both studies ranged from 50 to 5000 µg/plate.

Control plates without mutagen showed that the number of spontaneous revertant colonies was within the laboratory's historical control range. All the positive control compounds gave the expected increase in the number of revertant colonies.

Toxicity: In the mutagenicity experiments toxicity was not observed either with or without metabolic activation in a dose range of 4 to 5000 µg/plate.

Plate incorporation test: In the plate incorporation test the test substance did not result in relevant increases in the number of revertants in any of the bacterial strains both in the absence and in the presence of rat metabolic activation.

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 increase in the number of revertant colonies with any of the tester strains.

Under the test conditions, the test substance is not mutagenic in the standard plate test (Ames Test) and in the preincubation method according to

Prival.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The substance did not show any genotoxic effects in the in-vivo micronleus test 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 Feb. 16, 1998 to Jun. 05, 1998

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:

EU Method B.12 (Mutagenicity - In Vivo 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

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 Gartenstr. 27, D-33178 Borchen
- Age at study initiation: Approximately 7 wk
- Weight at study initiation: 34.9 g males; 29 g females
- Housing: Macrolon cages (type 3) on soft wood granulate in groups of 5 animals
- Diet: ssniff R/M-H (V1534), ad libitum
- Water: Tap water in plastic bottles, ad libitum
- Acclimation period: At least 7 d

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

IN-LIFE DATES: From Feb. 16, 1998 to May. 15, 1998

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Deionized water
- Justification for choice of solvent/vehicle: Soluble in deionized water
- Concentration of test material in vehicle: 2, 6, 20 %
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: On the days of administration the test substance was suspended in deionized water at the appropriate concentration (2, 6 and 20 %). A magnetic stirrer was used to keep the preparation homogeneous until dosing had been completed.

Duration of treatment / exposure:

48 h

Frequency of treatment:

Twice at an interval of 24 h

Post exposure period:

24 h after last dosing

Dose / conc.:

200 mg/kg bw/day (nominal)

Dose / conc.:

600 mg/kg bw/day (nominal)

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5/sex/dose

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Route of administration: Oral
- Doses / concentrations: 50 mg/kg bw and 0.5 % (w/v) concentration

Tissues and cell types examined:

Bone marrow erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Based on the results of a dose range finding assay

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Twice at an interval of 24 h and animals were killed by carbon dioxide asphyxiation 24 h after dosing

DETAILS OF SLIDE PREPARATION: For each animal, about 3 mL 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 min at approx, 1200 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.The specimens were fixed in methanol and stained with May-Grunwald/Giemsa.



METHOD OF ANALYSIS: Prior to microscopic assessment, all slides were furnished with code numbers, so that the counting was blind. The following counts were made:
Number of polychromatic erythrocytes (PCE) with micronuclei per 2000 erythrocytes
Number of micronuclei (MN) in 2000 erythrocytes.
Ratio of polychromatic erythrocytes to 200 total erythrocytes

Evaluation criteria:

A substance is considered positive if there is a significant increase in the number of micronucleated polychromatic erythrocytes compared with the concurrent negative control group.

Statistics:

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%

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: LD50>2000 mg/kg bw


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): No significant difference as compared to controls.
- Ratio of PCE/NCE (for Micronucleus assay): The ratio of PCE to total erythrocytes remained essentially unaffected by the test substance and was not less than 20 % of the control values
- Appropriateness of dose levels and route: Yes
- Statistical evaluation: Yes

Cyclophosphamide induced a marked and statistically significant increase in the number of polychromatic: erythrocytes with micronuclei.

None

Conclusions:

Under the test conditions, the test substance is considered not mutagenic in the micronucleus test in mice.

Executive summary:

A study was conducted to assess the potential of the test substance to cause chromosomal damage (clastogenicity) in a mouse bone marrow micronucleus test according to OECD Guideline 474, EPA OPPTS 870.5395 and EU method B.12. in compliance with GLP.

The test substance was suspended in deionized water and was given once as an orally dose of 200, 600, 2000 mg/kg bw to male and female mice (HsdWin:NMRI), based on the results of a dose range finding assay. At study start the animals were 7 wk of age and had mean body weights of 34.9 g (M) and 29 g (F). According to the test procedure the animals were killed 24 or 48 h after administration.

Cyclophosphamide was used as positive control substance and was administered once orally at a dose of 50 mg/kg bw.

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 by the treatment with test substance and was not less than 20 % of the control value.

Cyclophosphamide induced a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity of the test system. The ratio of polychromatic erythrocytes to total erythrocytes was not changed to a significant extent.

Test substance did not lead to a substantial increase of micronucleated polychromatic erythrocytes and is not mutagenic in the micronucleus test.

Under the test conditions, the test substance is considered not mutagenic in the micronucleus test in mice.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

The positive effect in the in-vitro chromosome aberration assay in V79 cells is considered to be a false positive result. Kirkland et al (2005) demonstrated an extremely high false-positive rate for in-vitro clastogenicity tests, particularly in mammalian cell tests, when compared to rodent carcinogenicity study results (Poth, A (2008)). “Certain characteristics of the commonly used rodent cell lines (CHO, CHL, V79, L5178Y, etc.), such as their p53 status, their karyotypic instability and their DNA repair deficiencies, are recognized as possibly contributing to the high rate of irrelevant positives. Also the need for exogenous metabolism with the cell systems is expected to contribute to these irrelevant positive findings, as metabolites produced by S9 used as a metabolic source in cell culture may be quite different from those produced by normal human liver metabolism.” Accordingly, “the high false-positive rate of the established in-vitro mammalian cell tests means that an increased number of compounds are subjected to earlier and additional in-vivo genotoxicity testing.”

In addition, it is well known that vinyl-sulphone compounds result in false positive test results in in-vitro tests for clastogenicity (Dearfield KL et al. (1991); Warra TJ et al. (1990)). This is due to the fact that these chemical agents react via the Michael addition reaction. Chemical reactivity via Michael addition is essential for many of the uses for which these compounds are important. Like in the currently assessed dye, vinyl sulphone moieties are used in fiber-reactive dyes (MacGregor et at. (1980)). These compounds are known to deplete glutathione in in‑vitro test systems, in which the concentration of phase II enzymes is very low. Glutathione plays a role in the detoxification of many compounds. Conjugation with glutathione via Michael addition and subsequent excretion is the most common bio-elimination route for these compounds. Since in-vitro systems have low levels of glutathione, the glutathione depletion leads to a positive result in the in-vitro test system, which is not the case in the in-vivo test system, where glutathione is present in adequate amount, as could be shown in the presen in vivo MNT study. Hence, the in-vivo test produces more reliable data for this kind of substance and the substance is not considered to have cytogenic effects.

 

References

Dearfield KL et al. (1991). Genotoxicity in mouse lymphoma cells .of chemicals capable of Michael addition. Mutagenesis 1991;6(6):519-525

Kirkland et al (2005). Evaluation of the ability of a battery of three in-vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens. I. Sensitivity, specificity and relative predictivity. Mutat Res. 2005 July 4;584(1–2):1–256

MacGregor et at. (1980). Mutagenicity tests of fabric-finishing agents in Salmonella typhimurium: fiber-reactive wool dyes and cotton flame retardants. Environ. Mutagenesis 1980;2:405-418

Poth, A (2008). Challenges in Testing for Genotoxycity. Genetic Toxicology and Cell Biology, RCC Cytotest Cell Research GmbH

Rothfuss A et al. (2011). Improvement of in-vivo genotoxicity assessment: combination of acute tests and integration into standard toxicity testing. Mutat Res. 2011 Aug 16;723(2):108-20.

Warra TJ et al. (1990). Methyl vinyl sulphone: A new class of Michael-type genotoxin. Mutat Res. 1990;245:191-199

Additional information

Mutagenicity:

in-vitro

A study was conducted to determine the mutagenic potential of test substance according to OECD Guideline 471, EPA OPPTS 798.5265 and EU method B.13/14.

Strains TA 100, TA 1535, TA 1537 and TA 98 of Salmonella typhimurium were used in the mutagenicity assay.

Two independent mutagenicity studies were conducted, one as the standard plate test 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 a rat liver homogenate or a hamster liver homogenate. For both studies, the compound was dissolved in aqua bidest., and each bacterial strain was exposed to 6 dose levels. Doses for both studies ranged from 50 to 5000 µg/plate.

Control plates without mutagen showed that the number of spontaneous revertant colonies was within the laboratory's historical control range. All the positive control compounds gave the expected increase in the number of revertant colonies.

Toxicity: In the mutagenicity experiments toxicity was not observed either with or without metabolic activation in a dose range of 4 to 5000 µg/plate.

Plate incorporation test: In the plate incorporation test the test substance did not result in relevant increases in the number of revertants in any of the bacterial strains both in the absence and in the presence of rat metabolic activation.

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 increase in the number of revertant colonies with any of the tester strains.

Under the test conditions, the test substance is not mutagenic in the standard plate test (Ames Test) and in the preincubation method according to Prival.

 

A structural analogue was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537 and TA 98 of Salmonella typhimurium and Escherichia coli WP2uvrA. Two independent mutagenicity studies were conducted one standard plate test ( Ames Test) and a modified preincubation test (Prival Test). The studies were performed in the absence and in the presence of a metabolizing system derived from a rat liver homogenate or a hamster liver homogenate. Additionally, a repeat of the preincubation test was performed with the strain WP2uvrA in the absence of S9-mix. For all studies, the compound was dissolved in deionized water, and each bacterial strain was exposed to 5 dose levels. Doses for all studies ranged from 50 to 5000 µg/plate. Control plates without mutagen showed that the number of spontaneous revertant colonies was within the laboratory's historical control range and similar to that described in the literature. All the positive control compounds gave the expected increase in the number of revertant colonies, except in the first preincubation test with the strain WP2uvrA in the absence of S9-mix, where the positive control showed not the expected increase in the number of revertant colonies. This questionable effect was caused by a decomposition of N-Methyl-N-nitrosoguanidine (MNNG). In a repeat of this strain with an alternative positive compound 4 -Nitroquinoline-N-Oxide (4 -NQO) the sensitivity of the assay could be demonstrated.

In the plate incorporation test and also in the preincubation test toxicity was not observed either with or without metabolic activation. In the standard plate test, the test substance did not result in relevant increases in the number of revertants in any of the bacterial strains in the absence or in the presence of the rat liver activation system (10 % (v/v)). Also 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 increases in the number of revertant colonies with any of the tester strains.

Therefore, the test substance is not mutagenic in the standard plate test (Ames Test) as well as in the preincubation method according to Prival at the investigated dose levels.

 

 

Clastogenicity

 

 in-vivo

 

A study was conducted to assess the potential of the test substance to cause chromosomal damage (clastogenicity) in a mouse bone marrow micronucleus test according to OECD Guideline 474, EPA OPPTS 870.5395 and EU method B.12.

The test substance was suspended in deionized water and was given once as an orally dose of 200, 600, 2000 mg/kg bw to male and female mice (HsdWin:NMRI), based on the results of a dose range finding assay. At study start the animals were 7 wk of age and had mean body weights of 34.9 g (M) and 29 g (F). According to the test procedure the animals were killed 24 or 48 h after administration.

Cyclophosphamide was used as positive control substance and was administered once orally at a dose of 50 mg/kg bw.

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 by the treatment with test substance and was not less than 20 % of the control value.

Cyclophosphamide induced a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity of the test system. The ratio of polychromatic erythrocytes to total erythrocytes was not changed to a significant extent.

 

Test substance did not lead to a substantial increase of micronucleated polychromatic erythrocytes and is not mutagenic in the micronucleus test.

The test substance is considered not mutagenic in the micronucleus test in mice (Dr. Muller W, 1998)

 

in-vitro

A study was conducted to investigate the potential of test substance to induce chromosome aberrations in V 79 cells of the Chinese hamster lung in vitro according to OECD Guideline 473, EPA OPPTS 870.5375 and EU Method B.10.

The test substance was dissolved in DMSO and tested at the following concentrations:

First experiment with 20 h treatment time:

without S9-mix: 10, 25, 50, 100, 250 and 500 µg/mL

with S9-mix: 100, 250, 500, 1000 and 1500 µg/mL

Second experiment with 28 h treatment time:

without S9-mix: 10, 25, 50, 100, 250 and 500 µg/mL

with S9-mix: 100, 250, 500, 1000 and 1500 µg/mL

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.

In the highest investigated dose the test compound induced a significant increase in the number of chromosome aberrations in the presence and in the absence of metabolic activation.

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.

Test substance does induce chromosome aberrations in V79 Chinese hamster cells, both in the presence as well as in the absence of a metabolic activation system.

The test substance was clastogenic in the in vitro chromosome aberration assay with V79 Chinese hamster lung cells (Dr. Muller W, 1998)

Justification for classification or non-classification

The test substance was found to be non-genotoxic both in in-vitro bacterial reverse mutation and in-vivo micronucleus test in mice. However, it was positive for genotoxicity in in-vitro chromosomal aberration chromosomal aberration test.

Based on the overall weight of evidence test substance, is expected not to have any genotoxic potential. Therefore no classification is required for genotoxicity according to EC criteria (67/548/EEC) and according to CLP criteria (EC 1272/2008).