Reaction mass of lithium sodium hydrogen 4-amino-6-({5-[(5-chloro-2,6-difluoropyrimidin-4-yl)amino]-2-sulfonatophenyl}diazenyl)-5-hydroxy-3-[(4-{[2-(sulfonatooxy)ethyl]sulfonyl}phenyl)diazenyl]naphthalene-2,7-disulfonate and lithium sodium hydrogen 4-amino-6-({5-[(5-chloro-2,6-difluoropyrimidin-4-yl)amino]-2-sulfonatophenyl}diazenyl)-5-hydroxy-3-{[4-(vinylsulfonyl)phenyl]diazenyl}naphthalene-2,7-disulfonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

other: Ames et al. 1973, 1975

Principles of method if other than guideline:

The test method followed the procedure described at Ames et al, 1973 and Ames et al, 1975.

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

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from rat liver

Test concentrations with justification for top dose:

First plate incorporation test, with and without S9 mix:
0 (negative control), 16, 80, 400, 2000 and 10,000 µg/plate
Positive control:
sodium azide: 10 µg/plate (only TA 1535)
nitrofurantoin: 0.2 µg/plate (only TA 100)
4-nitro-o-phenylendiamine: 10 µg/plate (only TA 1537)
4-nitro-o-phenylendiamine: 0.5 µg/plate (only TA 98)
2-aminoanthracene: 3 µg/plate

Second plate incorporation test, with and without S9 mix:
The doses of test substance were increased to 625 - 10,000 µg/plate as no toxicity was observed.

Vehicle / solvent:

Deionized water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Only for TA 1535

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: Nitrofurantoine

Remarks:

Only for TA 100

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylendiamine

Remarks:

Only for TA 1537 and TA 98

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Evaluation criteria:

Criteria for a positive response
A response is considered positive if there is a reproducible dose-dependent increase in the number of mutants in at least one strain, with numbers reaching approximately the double of those of the negative control.

Key result

Species / strain:

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Conclusions:

Under the study conditions, the test substance was not found to be mutagenic in the bacterial reverse mutation assay.

Executive summary:

An in vitro study was performed to investigate the potential of the test substance to induce gene mutations according to the method of Ames (1973, 1975), in compliance with GLP. Two independent assays were conducted, using the plate incorporation method, in the absence and presence of a metabolizing system derived from a rat liver homogenate (i.e. S9 mix). The substance was tested at five concentrations in the range of 16 - 10,000 µg/plate in the first assay and, as no toxicity occurred, at 625 - 10,000 µg/plate in the second assay. The tested strains were Salmonella typhimurium TA 1535, TA 1537, TA 98 and TA 100. Doses up to and including 10,000 µg/plate did not induce any bacteriotoxic effects. The total bacteria counts remained unchanged. No inhibition of growth was observed. Evidence of mutagenic activity of the test substance was not found. Neither a dose-related doubling of mutant counts nor a biologically relevant increase in the same, in comparison with the negative controls, was seen. The positive controls had a marked mutagenic effect. Under the study conditions, the test substance was not found to be mutagenic in the bacterial reverse mutation assay (Herbold, 1986).

Reference 2

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From March 20, 2001 to May 16, 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.18 (DNA Damage and Repair - Unscheduled DNA Synthesis - Mammalian Cells In Vitro)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5550 - Unscheduled DNA Synthesis in Mammalian Cells in Culture

Deviations:

no

GLP compliance:

yes

Type of assay:

DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro

Species / strain / cell type:

hepatocytes:

Details on mammalian cell type (if applicable):

-Origin (supplier) of animals: Harlan Winkelmann GmbH Gartenstrasse 27,33178 Borchen
-Source of biological material: Hepatocyte cells obtained from rats
-Cell culture medium: Hepatocyte Attachment Medium (mfr. Gibco BRL) without fibronectin
-Experimental conditions in vitro: approximately 37°C and approximately 5% CO2 in 35 mm culture dishes

Metabolic activation:

not applicable

Test concentrations with justification for top dose:

0, 0.1, 0.3, 1, 3, 10, 30, 100, 300, 1000, 3000 and 5000 µg/mL

Vehicle / solvent:

Cell culture medium

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

Details on test system and experimental conditions:

Experimental design

- Two independent assays for the unscheduled DNA synthesis test in primary rat hepatocytes were performed. A dose range of seven concentrations from 0.1 to 100 µg/mL was employed in both mutation assays.

Test Procedure (i.e. Unscheduled DNA synthesis test)

- Approximately 1.5 x 10E+5 cells were seeded in a 35 mm culture dish, which contained a coverslip treated previously with collagen. The cells were allowed to attach with approximately 5% CO2 for approximately 2 h at 37°C. Two culture dishes were used for each experimental point.

- The test substance was dissolved in cell culture medium. The incubation of the test and control substances at various concentrations was performed at 37°C for 16 - 20 h.

- Tritiated thymidine was added to the cell culture immediately after introduction of the test substance.

- At the end of the treatment the incubation medium was removed and the cells were rinsed twice. In order to get a better grain quantification by enlarging the nucleus, the cells were treated with 1% sodium citrate. After this they were mounted with ethanol: glacial acetic acid 3:1. After the coverslips had been mounted, they were rinsed with double distilled water and dried. Then they were glued to a slide, covered with Kodak NTB 2 autoradiographic film emulsion and stored for 4 to 6 d in a light-proof box, containing a drying agent, in the refrigerator. After this they were developed with Kodak D 19 developer and stained with the modified hematoxylin-eosin method.

- The incorporation of [3H] thymidine was determined by counting the number of grains in the nucleus and the number of grains in a nucleus sized area in the cytoplasm. The difference between these examination points is expressed as net grains per nucleus. Heavily labeled S-phase cells were not included in the count. 50 cells were evaluated per slide.

Evaluation criteria:

Criteria for a positive response

For the evaluation of the data the following criteria were used.

- If the net nuclear grain number was at least five grains higher than the corresponding solvent control value, the results for that dose were considered significant.

- The test substance is classified as genotoxic if it reproducibly induces with one of the test substance concentrations a significant increase in [3H] thymidine incorporation in comparision with the solvent control.

- The test substance is classified as genotoxic if there is a reproducible significant concentration related increase in [3H] thymidine incorporation in comparision with the solvent control.

- In the final instance, however the interpretation of the results were based on scientific judgement.

Key result

Species / strain:

hepatocytes:

Metabolic activation:

not applicable

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at the four highest concentrations (i.e. ≥300 µg/mL)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- The test substance was assessed for its genotoxic potential in vitro in the UDS - test in two independent experiments. No relevant reproducible increase in incorporation of [3H] thymidine over the range of the negative control was found with any of the test concentrations used.

- The sensitivity of the test system is demonstrated by the increased incorporation of tritiated thymidine in the cell cultures treated with the positive control substance.

Remarks on result:

other: all strains/cell types tested

Solubility and toxicity

 

In a preliminary experiment, the test substance was tested with respect to its solubility in hepatocyte attachment medium. The highest concentration at which no visible precipitation was observed, was found to be 5,000 µg/mL.

 

Based on these results 5,000 µg/mL was determined as maximum dose level for the main assays and ten lower concentrations were included in the treatment series.

Conclusions:

Under the study conditions, the test substance did not induce DNA damage in primary rat hepatocytes.

Executive summary:

A study was conducted to investigate the potential of the test substance to induce DNA repair in primary hepatocytes of male rats according to OECD Guideline 482, EU Method B.18 and EPA OPPTS 870.5550 Method, in compliance with GLP. Two independent assays were conducted, with incubation time of 16 - 20 h at 37°C. The test substance was dissolved in cell culture medium (i.e. hepatocyte attachment medium without fibronectin) and tested at the concentrations ranging from 0.1-5,000 µg/mL, based on the results of preliminary testing for solubility and toxicity. Positive controls showed a distinct increase in incorporation of [³H] thymidine in the cells, thus indicating the sensitivity of the assay. The test substance did not induce a relevant reproducible increase in the incorporation of [³H] thymidine in the cells up to the highest evaluated concentration of 100 µg/mL in either of the experiments. Further, as a sign of cytotoxic effects, the hepatocyte nuclei of the four highest concentrations (i.e. ≥300 µg/mL) showed incorporation of the test substance indicating a strongly decreased vitality. Therefore, these concentrations were not evaluated. Under the study conditions, the test substance did not induce DNA damage in primary rat hepatocytes (Kauffmann, 2001).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Principles of method if other than guideline:

The test was conducted according to the method published by Salamone et al., 1980.

Salamone M, Heddle J, Stuart E and Katz M, 1980. Towards and improved micronucleus test. Studies on 3 model agents, mitomycin C, cyclophosphamide and dimethylbenzanthracene. Mutat. Res. 74:347-356.

GLP compliance:

yes

Type of assay:

other: Mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: F. Winkelmann, Borchen
- Age at study initiation: 8 to 12 wk
- Weight at study initiation: 23-34 g
- Housing: Makrolon Type II cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum):ad libitum
- Acclimation period: one wk

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-22°C
- Humidity (%): 44-50%
- Photoperiod (hrs dark / hrs light): 12:12

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 0.5% aqueous cremophor emulsion
- Justification for choice of solvent/vehicle: the test substance has a better dispersibility in this vehicle
- Amount of vehicle (if gavage or dermal): 20 mL/kg bw

Duration of treatment / exposure:

24, 48 and 72 h for three separate test groups dosed at 7500 mg/kg bw
24 h for negative and positive control groups

Frequency of treatment:

Once

Dose / conc.:

7 500 mg/kg bw (total dose)

No. of animals per sex per dose:

Ten (five males and five females) per dose group including negative and positive control groups

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Route of administration: Oral gavage
- Doses / concentrations: 20 mg/kg bw
- Dose volume: 10 mL/kg bw

Tissues and cell types examined:

Bone marrow from femur was obtained and the polychromatic erythrocytes were examined.

Details of tissue and slide preparation:

TREATMENT AND SAMPLING TIMES:
Three groups of mice were treated with the test substance and were sacrificed at 24 h interval (i.e., at 24, 48 and 72 h) to obtain the bone marrow samples.

DETAILS OF SLIDE PREPARATION:
The bone marrow preparation was stained with Ames Hema-Tek slide stainer, followed by treatment with methanol and washing with demineralised water. Once the slides are dried, they ere observed under microscope.

METHOD OF ANALYSIS: 1000 polychromatic erythrocytes were examined per animal and the frequency of micronucleated cells among them were determined.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- Following treatment with the test substance, no relevant increase in the number of micronucleated PCEs over the concurrent negative control was observed at the treated dose and at all the exposure intervals. A marked increase in the frequency of micronucleated PCEs was noticed in the positive control group.
- Bone marrow cell toxicity: not specified

Conclusions:

Under the study conditions, the test substance did not induce micronuclei in the polychromatic erythrocytes of the treated mice.

Executive summary:

A study was conducted to determine the in vivo genetic toxicity of the test substance in NMRI mice according to the method published by Solomone et al, 1980. The ability of the test substance to induce cytogenetic damage and/or disruption of the mitotic apparatus in rat bone marrow was investigated by measuring the induction of micronuclei in polychromatic erythrocytes. NMRI mice (5 per sex per group) were exposed to a single oral gavage dose of the test substance at 0 and 7500 mg/kg bw for 24, 48 and 72 h. A positive control group (cyclophosphamide, 20 mg/kg bw) was also tested. Following treatment with the test substance, no relevant increase in the number of micronucleated PCEs over the concurrent negative control was observed at any of the exposure interval. A marked increase in the frequency of micronucleated PCEs was noticed in the positive control group. The values for the positive and negative controls were within the expectation ranges. The experiment was therefore considered valid. Under the study conditions, the test substance did not induce micronuclei in the polychromatic erythrocytes of the treated mice (Herbold, 1986b).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

No genotoxic activity of the test substance was observed in the in vitro and in vivo studies.

Additional information

In vitro

An in vitro study was performed to investigate the potential of the test substance to induce gene mutations according to the method of Ames (1973, 1975), in compliance with GLP. Two independent assays were conducted, using the plate incorporation method, in the absence and presence of a metabolizing system derived from a rat liver homogenate (i.e. S9 mix). The substance was tested at five concentrations in the range of 16 - 10,000 µg/plate in the first assay and, as no toxicity occurred, at 625 - 10,000 µg/plate in the second assay.The tested strains were Salmonella typhimurium TA 1535, TA 1537, TA 98 and TA 100. Doses up to and including 10,000 µg/plate did not induce any bacteriotoxic effects. The total bacteria counts remained unchanged. No inhibition of growth was observed. Evidence of mutagenic activity of the test substance was not found. Neither a dose-related doubling of mutant counts nor a biologically relevant increase in the same, in comparison with the negative controls, was seen. The positive controls had a marked mutagenic effect. Under the study conditions, the test substance was found not to be mutagenic in the bacterial reverse mutation assay (Herbold, 1986a).

A study was conducted to investigate the potential of the test substance to induce DNA repair in primary hepatocytes of male rats according to OECD Guideline 482, EU Method B.18 and EPA OPPTS 870.5550 Method, in compliance with GLP.Two independent assays were conducted, with incubation time of 16 - 20 h at 37°C. The test substance was dissolved in cell culture medium (i.e. hepatocyte attachment medium without fibronectin) and tested at the concentrations ranging from 0.1-5,000 µg/mL, based on the results of preliminary testing for solubility and toxicity. Positive controls showed a distinct increase in incorporation of [³H] thymidine in the cells, thus indicating the sensitivity of the assay. The test substance did not induce a relevant reproducible increase in the incorporation of [³H] thymidine in the cells up to the highest evaluated concentration of 100 µg/mL in either of the experiments. Further, as a sign of cytotoxic effects, the hepatocyte nuclei of the four highest concentrations (i.e. ≥300 µg/mL) showed incorporation of the test substance indicating a strongly decreased vitality. Therefore, these concentrations were not evaluated. Under the study conditions, the test substance did not induce DNA damage in primary rat hepatocytes (Kauffmann, 2001).

In vivo

A study was conducted to determine the in vivo genetic toxicity of the test substance in NMRI mice according to the method published by Solomone et al., 1980. The ability of the test substance to induce cytogenetic damage and/or disruption of the mitotic apparatus in rat bone marrow was investigated measuring the induction of micronuclei in polychromatic erythrocytes. NMRI mice (5 per sex per group) were exposed to a single oral gavage dose of the test substance at 0 and 7500 mg/kg bw for 24, 48 and 72 h. A positive control group (cyclophosphamide, 20 mg/kg bw) was also tested. Following treatment with the test substance, no relevant increase in the number of micronucleated PCEs over the concurrent negative control was observed at any of the exposure interval. A marked increase in the frequency of micronucleated PCEs was noticed in the positive control group. The values for the positive and negative controls were within the expectation ranges. The experiment was therefore considered valid. Under the study conditions, the test substance did not induce micronuclei in the polychromatic erythrocytes of the treated mice (Herbold, 1986b).

Justification for classification or non-classification

Based on the in vitro and in vivo test results, the test substance warrants no classification for genotoxicity according to CLP (EC/1272/2008) criteria.