1,7-Naphthalenedisulfonic acid, 2-[[4-chloro-6-(cyanoamino)-1,3,5-triazin-2-yl]amino]-5-hydroxy-6-[2-[2-methoxy-5-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]diazenyl]-, lithium sodium salt (1:?:?)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A bacterial reverse mutation test including the Prival modification in strains TA 100, TA 1535, TA 1537 and TA 98 of Salmonella typhimurium and Escherichia coli WP2uvrA showed an increased mutation frequency in the absence of exogenous metabolic activation in the preincubation test in S. typhimurium TA 98 and TA 1537. However, the substance was negative in an in-vitro mutagenicity study in mammalian cells (Chinese hamster V79 HPRT-assay). The substance showed a false positive effect in an in-vitro chromosome aberration assay in V79 cells without metabolic activation but was negative in an in-vivo micronucleus test in mice. Weight of evidence based on available data (HPRT negative; MNT in-vivo negative) indicate that the substance is not mutagenic. 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From Nov. 17, 1999 to Apr. 10, 2000

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:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EEC Directive 87/302, L133, pp. 61 - 63, March 1987

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

HRPT locus (V79 Chinese hamster cells)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Source: cell bank of "Genetic Toxicology", Aventis Pharma Deutschland GmbH, ProTox
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

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Main mutation experiment (First):
- without S9-mix: 100, 250, 500, 1000, 1750, 2500, 3750 and 5000* µg/mL
- with S9-mix: 100, 250, 500, 1000, 1750, 2500, 3750 and 5000 µg/mL

* = because of high toxicity in the first main experiment no mutant selection was performed

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9,10-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: Seeded in each well of a microtiter plate

DURATION (Preliminary toxicity)
- Preincubation period: 12 h (overnight)
- Exposure duration: 4 h
- Incubation period: 24 h
- Fixation and staining: Crystal violet
NUMBER OF REPLICATIONS: 6 (For each concentration at least 6 wells)

DURATION (Main experiment)
- Preincubation period: 24 h
- Exposure duration: 4 h (Medium+ test substance+ buffer/S9-mix)
- Incubation period: 24 h
- Fixation and staining: Crystal violet

Evaluation criteria:

The test item was considered positive if
(a) It reproducibly induces with one of the test compound concentrations a mutation frequency that is three times higher than the spontaneous mutant frequency in this experiment.
(b) 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.
(c) Survival of the responding dose group is at least 30 %.
However, in a case by case evaluation both decisions depend on the level of the corresponding negative control data.

Statistics:

- The biometry of the results for the test compound is performed off-line with the Mann-Whitney- U-Test

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

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 concentration at which no visible precipitation was observed.

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.

Based on the preliminary study results, 5000 µg/mL was selected as the maximum dose level for the main mutation experiments in both the absence and in the presence of S9-mix. Seven lower concentrations down to 100 µg/mL were also included.

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:

Main experiment: A significant increase of the mutation frequency was observed in the presence of metabolic activation at the concentrations of 250, 2500 and 3750 µg/mL.

These results were not reproducible and not three fold higher than the corresponding controls and were therefore considered to be of no biological relevance.

No relevant reproducible increase in the mutant colonies or mutant frequency over the range of the solvent control was found with any other concentration, either with or without metabolic activation by S9-mix.

Conclusions:

The test substance is considered to be non-mutagenic in the HPRT-test with V79 Chinese hamster cells, either in the presence or in the absence of metabolic activation.

Executive summary:

The study was performed to investigate the potential of test substance to induce gene mutations at the HPRT locus in V 79 cells of the Chinese hamster in vitro according to OECD Guideline 476, EPA OPPTS 870.5300 and EEC Directive 87/302, L133 in compliance with GLP.

Two independent experiments were conducted both with and without an exogenous rat liver microsomal activation system (S9-mix). The test substance was dissolved in cell culture medium and tested at the following concentrations:

- without S9-mix: 100, 250, 500, 1000, 1750, 2500, 3750 and 5000 µg/mL

- with S9-mix: 100, 250, 500, 1000, 1750, 2500, 3750 and 5000 µg/mL

Due to the limitation of the OECD guideline 5000 µg/mL is the highest tolerated dose.

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

Test substance produced no macroscopical precipitation. Up to the highest investigated dose no relevant and reproducible increase in mutant colony numbers was obtained in two independent experiments.

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

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

Under the test conditions, the test substance is considered to be non-mutagenic in the HPRT-test with V79 Chinese hamster cells, either in the presence or absence of metabolic activation.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From Aug. 10, 1999 to Nov. 18, 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

1998

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1997

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", Aventis Pharma Deutschland GmbH, ProTox
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):

Colcemid

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

First experiment with 3 h treatment time:
without S9-mix: 0, 500, 1000, 2500, 3750$ and 5000 µg/mL
with S9-mix: 500#, 1000, 2500 and 5000 µg/mL

# not used because higher concentrations were evaluated
$ additionally evaluated concentration

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

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)
STAIN (for cytogenetic assays): 2 % (w/v) orcein solution

NUMBER OF REPLICATIONS: Two

NUMBER OF CELLS EVALUATED: 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 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 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:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

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 practicable concentration and produced no macroscopical precipitation.
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 for 3 h in the absence and in the presence of S9 metabolic activation, toxicity was observed at 5000 µg/mL. Survival declined in a dose-related manner reaching 59.2 % of the solvent control value at the highest dose level, 5000 µg/mL, in the absence of S9-mix. In the presence of S9 metabolic activation at a concentration of 5000 µg/mL survival was reduced to 55.5 %of the solvent control value. After 20 h treatment survival was reduced to 51.8 % of the solvent control value at a concentration of 1000 µg/mL

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 main experiment the cytotoxicity was also evaluated by treatment of cells seeded in microwell plates. Survival was reduced in a dose-related manner reaching 68.7% of the solvent control value at the highest concentration without S9-mix. With S9-mix survival was reduced in a dose-related manner reaching 65.6 % of the solvent control value at 5000 µg/mL at the 3 h treatment time.

In the first main experiment the mitotic index was reduced (indication of toxicity) after treatment with the highest dose levels. After treatment with the test substance there was no relevant increase in the number of polyploid cells as compared with the solvent controls.

There was a statistically significant increase of the aberration rates at the 3 h treatment time with 3750 and 5000 µg/mL without S9-mix. On the basis of these findings the evaluation of slides of the longer treatment time (20 h) was not necessary. The sensitivity of the test system was demonstrated by the enhanced mutation frequency in the cell cultures treated with the positive control compounds.

Conclusions:

The test substance was clastogenic in the in vitro chromosome aberration assay with V79 Chinese hamster lung cells without metabolic activation.

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 lungin 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 cell culture medium and tested at the following concentrations:

First experiment with 3 h treatment time:

without S9-mix: 1000, 2500, 3750 and 5000 µg/mL

with S9-mix: 1000, 2500 and 5000 µg/mL

Due to the limitation of the OECD guideline 5000 µg/mL is the highest tolerated dose.

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

Precipitation of the test compound was not observed.

In the absence of metabolic activation the test compound induced statistically significant increases in the number of phases with chromosome aberrations at the concentrations of 3750 and 5000 µg/mL.

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, 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 Jun. 30, 1999 to Nov. 26, 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

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from induced rat liver and uninduced hamster liver

Test concentrations with justification for top dose:

Plate incorporation test:
a: without metabolic activation:
50, 160, 500, 1600 and 5000 µg/plate
b: with metabolic activation:
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:
50, 160, 500, 1600 and 5000 µg /plate

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

congo red

other: 2-aminoanthracene

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:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

plate incorporation

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

Genotoxicity:

negative

Remarks:

preincubation

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:

without

Genotoxicity:

positive

Remarks:

preincubation

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

Remarks:

plate incorporation

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

Genotoxicity:

negative

Remarks:

preincubation

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:

without

Genotoxicity:

positive

Remarks:

preincubation

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 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

plate incorporation and preincubation

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

Remarks:

plate incorporation and preincubation

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

Remarks:

plate incorporation and preincubation

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.

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 the Salmonella strains TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli WP2uvrA in the absence and in the presence of rat metabolic activation.

Preincubation test:

In the absence of the metabolic activation systems the test compound induced a significant and dose-dependent increase in the number of revertant colonies with the bacterial strains TA 1537 and TA 98 in the preincubation test.

In the presence, of hamster liver 59-mix (30 % (v/v)) using the preincubation method according to Prival the test compound 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 considered to be mutagenic in the absence of exogenous metabolic activation in the preincubation test in S. typhimurium TA 98 and TA 1537.

Executive summary:

A study was conducted to determine the mutagenic potential of test substance according to OECD Guideline 471, EPA OPPTS 870.5100 and EU method B.13/14. in compliance with GLP.

Strains TA 100, TA 1535, TA 1537 and TA 98 of Salmonella typhimurium and Escherichia coli WP2uvrA 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 deionized water, and each bacterial strain was exposed to 5 dose levels.

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

Plate incorporation test: In the plate incorporation test the test substance did not cause a significant increase in the number of revertant colonies with the Salmonella strains TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli WP2uvrA in the absence and in the presence of rat metabolic activation.

Preincubation test: In the absence of the metabolic activation systems the test substance induced a significant and dose-dependent increase in the number of revertant colonies with the bacterial strains TA 1537 and TA 98 in the 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 under the experimental conditions described.

Under the test conditions, the test substance is considered to be mutagenic in the absence of exogenous metabolic activation in the preincubation test in S. typhimurium TA 98 and TA 1537.

Genetic toxicity in vivo

Description of key information

The substance showed no cytogenic effects in a mouse micronucleus test

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 Feb. 08, 2000 to Mar. 24, 2000

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: 32.3 g males; 27.5 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 5 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. 08, 2000 to Feb. 23, 2000

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: 0, 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 (0 and 20 %). A magnetic stirrer was used to keep the preparation homogeneous until dosing had been completed.

Duration of treatment / exposure:

Not applicable

Frequency of treatment:

Twice at an interval of 24 h

Post exposure period:

24 h after last dosing

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.

OTHER:

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%

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

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

Conclusions:

The test substance is considered not cytogenic 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.2. in compliance with GLP.

The test substance was suspended in deionized water and was given twice at an interval of 24 h as an oral dose of 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 32.3 g (M) and 27.5 g (F). According to the test procedure the animals were killed 24 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.

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

In the chromosomal aberration test, the test compound induced an increase in the number of chromosome aberrations in the absence of S9-mix at the two highest concentrations at cytotoxic concentrations.

However, 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.”

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. As 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 plenty of tests with vinyl sulphone dyes (internal data DyStar). Hence, the in-vivo test, which was clearly negative, produces more reliable data for this kind of substance.

Additional information

Mutagenicity:

 

A study was performed to investigate the potential of test substance to induce gene mutations at the HPRT locus in V 79 cells of the Chinese hamster in vitro according to OECD Guideline 476, EPA OPPTS 870.5300 and EEC Directive 87/302, L133.

Two independent experiments were conducted both with and without an exogenous rat liver microsomal activation system (S9-mix). The test substance was dissolved in cell culture medium and tested at the following concentrations:

- without S9-mix: - without S9-mix: 100, 250, 500, 1000, 1750, 2500, 3750 and 5000 µg/mL

- with S9-mix: 100, 250, 500, 1000, 1750, 2500, 3750 and 5000 µg/mL

Due to the limitation of the OECD guideline 5000 µg/mL is the highest tolerated dose.

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

Test substance produced no macroscopical precipitation. Up to the highest investigated dose no relevant and reproducible increase in mutant colony numbers was obtained in two independent experiments.

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

Test substance does not induce gene mutations in the HPRT-test with V79 Chinese hamster cells, both in the presence as well as in the absence of a metabolic activation system. The test substance is considered to be non-mutagenic in the HPRT-test with V79 Chinese hamster cells, either in the presence or absence of metabolic activation (Dr. Stammberger I, 2000).

 

A study was conducted to determine the mutagenic potential of test substance according to OECD Guideline 471, EPA OPPTS 870.5100 and EU method B.13/14.

Strains TA 100, TA 1535, TA 1537 and TA 98 of Salmonella typhimurium and Escherichia coli WP2uvrA 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 deionized water, and each bacterial strain was exposed to 5 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.

Plate incorporation test: In the plate incorporation test the test substance did not cause a significant increase in the number of revertant colonies with the Salmonella strains TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli WP2uvrA in the absence and in the presence of rat metabolic activation.

Preincubation test: In the absence of the metabolic activation systems the test substance induced a significant and dose-dependent increase in the number of revertant colonies with the bacterial strains TA 1537 and TA 98 in the 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 under the experimental conditions described. The test substance is considered to be mutagenic in the absence of exogenous metabolic activation in the preincubation test (Dr. Stammberger I and Braun K DI, 1999).

Test substance did not cause a significant increase in the number of revertant colonies in the bacterial reverse mutation test (plate incorporation) at doses up to 5,000 µg/plate with the strains TA100, TA1535, TA1537, and TA98 of Salmonella typhimurium and WP2uvrA of Escherichia coli. Using the preincubation test without metabolic activation test substance was mutagenic. This is in contrast to the results of the preincubation test using hamster liver S9-mix whereas no significant increase in the number of revertant colonies occurred. 

 

Clastogenicity:

 

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.2.

The test substance was suspended in deionized water and was given twice at an interval of 24 h as an oral dose of 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 32.3 g (M) and 27.5 g (F). According to the test procedure the animals were killed 24 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. The test substance is considered not mutagenic in the micronucleus test in mice (Dr. Roth T, 2000).

 

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 cell culture medium and tested at the following concentrations:

First experiment with 3 h treatment time:

without S9-mix: 1000, 2500, 3750 and 5000 µg/mL

with S9-mix: 1000, 2500 and 5000 µg/mL

Due to the limitation of the OECD guideline 5000 µg/mL is the highest tolerated dose.

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

Precipitation of the test compound was not observed. In the absence of metabolic activation the test compound induced statistically significant increases in the number of phases with chromosome aberrations at the concentrations of 3750 and 5000 µg/mL. 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, 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. Stammberger I and Graser H DI, 1999).

Justification for classification or non-classification

The test substance was found to be non-genotoxic both in in-vitro HPRT and in-vivo micronucleus test in mice. However, it was positive for genotoxicity in in-vitro bacterial reverse mutation and chromosomal aberration tests, both without metabolic activation.

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).