Disodium hydrogen bis[3-hydroxy-4-[(2-hydroxy-1-naphthyl)azo]naphthalene-1-sulphonato(3-)]chromate(3-)

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 mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2012-06-13 to 2012-09-17

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP Guideline Study performed on the analogue substance

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany)

Type of assay:

mammalian cell gene mutation assay

Target gene:

hypoxanthine-guanine-phosphoribosyl-transferase (HPRT)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

-Type and identity of media: MEM- Properly maintained: yes- Periodically checked for Mycoplasma contamination: yes- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 of Sprague Dawley Phenobarbital and ß-Naphthoflavone-induced rat liver S9 mix

Test concentrations with justification for top dose:

All concentrations used referred to the active components of the test item.Pre-experiment for experiment I without metabolic activation: 3.16, 10.0, 31.6, 100, 316, 1000, 2500, 5000 µg/mLwith metabolic activation: 1.0, 2.5, 5.0, 10.0, 31.6, 100, 316, 1000, 2500, 5000 µg/mLExperiment Iwithout metabolic activation: 0.025, 0.05, 0.10, 0.25, 0.50, 1.0, 2.5, 5.0, 10 and 20 µg/mLwith metabolic activation: 10, 25, 50, 100, 150, 175, 200, 225, 250, and 275 µg/mLExperiment IIwithout metabolic activation: 10, 20, 40, 50, 60, 70, 80, 90 and 100 µg/mLwith metabolic activation: 30, 60, 140, 220, 240, 250, 260, 270, 280 and 290 µg/mL

Vehicle / solvent:

Vehicle (Solvent) used: cell culture medium (MEM + 0% FBS 4h treatment; MEM + 10% FBS 20h treatment). The test item was dissolved in cell culture medium and diluted prior to treatment

Untreated negative controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activationMigrated to IUCLID6: 300 µg/mL

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activationMigrated to IUCLID6: 1 µg/mL and 1.5 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: suspended in mediumDURATION: 4 h (short-term exposure), 20 h (long-term exposure)Expression time (cells in growth medium): 48-72 hSelection time (if incubation with selection agent): about one weekSELECTION AGENT ( mutation assay) 11 µg/mL 6-thioguanine (TG)NUMBER OF REPLICATIONS: two separate experiments (I+II) with single exposure; 5 individual flasks were seeded and evaluatedNUMBER OF CELLS EVALUATED: 400000 cells per flaskDETERMINATION OF CYTOTOXICITY: Method: relative growth

Evaluation criteria:

A test is considered to be negative if there is no biologically relevant increase in the number of mutants.There are several criteria for determining a positive result:-a reproducible three times higher mutation frequency than the solvent control for at least one of the concentrations; -a concentration related increase of the mutation frequency; such an evaluation may be considered also in the case that a three-fold increase of the mutant frequency is not observed;-if there is by chance a low spontaneous mutation rate in the corresponding negative and solvent controls a concentration related increase of the mutations within their range has to be discussed.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment I without S9: ≥ 5.0 μg/mL; experiment I with S9: ≥ 225 μg/mL; Experiment II without S9: ≥ 60 μg/mL; Experiment II with S9:≥ 220 μg/mL

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):negativeIn vitro cell gene mutagenicity test was performed on the analogue substance following OECD 476 (HPRT-test). Under the experimental conditions reported, the analogue substance is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.

Executive summary:

In a mammalian cell gene mutation assay (HPRT locus],V79 cells cultured in vitro were exposed to the analogue substance dissolved in cell culture medium (MEM + 0% FBS 4h treatment; MEM + 10% FBS 20h treatment) at concentrations of

- 0.025, 0.05, 0.10, 0.25, 0.50, 1.0, 2.5, 5.0, 10 and 20 µg/mL (without metabolic activation, Experiment I)

- 10, 25, 50, 100, 150, 175, 200, 225, 250 and 275 µg/mL (with metabolic activation, Experiment I)

- 10, 20, 40, 50, 60, 70, 80, 90 and 100 µg/mL (without metabolic activation, Experiment II)

- 30, 60, 140, 220, 240, 250, 260, 270, 280 and 290 µg/mL µg/mL (with metabolic activation, Experiment II).

Analogue substance was tested up to cytotoxic concentrations.

Biologically relevant growth inhibition was observed in experiment I and II with and without metabolic activation. In experiment I without metabolic activation the relative growth was 5.1% for the highest concentration (20 µg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 275 µg/mL with a relative growth of 19.5%. In experiment II without metabolic activation the relative growth was 20.1% for the highest concentration (100 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 290 µg/mL with a relative growth of 10.1%.

In experiment I without metabolic activation the highest mutation rate (compared to the negative control values) of 0.80 was found at a concentration of 0.025 µg/mL with a relative growth of 76.4%.

In experiment I with metabolic activation the highest mutation rate (compared to the negative control values) of 2.60 was found at a concentration of 250 µg/mL with a relative growth of 31.7%.
In experiment II without metabolic activation the highest mutation rate (compared to the negative control values) of 1.33 was found at a concentration of 100 µg/mL with a relative growth of 20.1%.
In experiment II with metabolic activation the highest mutation rate (compared to the negative control values) of 1.98 was found at a concentration of 140 µg/mL with a relative growth of 83.1%.

The positive controlsdidinduce the appropriate response. 

There was no evidence of a concentration related positive response of induced mutant colonies over background.

This study is classified as acceptable.  This study satisfies the requirement for Test Guideline OPPTS 870.5300, OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data.

 

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro cytogenicity study in mammalian cells or in vitro micronucleus study does not need to be conducted because adequate data from an in vivo cytogenicity test are available

Reference 3

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:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

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:

for plate incorporation Main Assa i: Rat liver S9 (standar metabolic activation)
for pre-imcubation Main Assay II: hamster liver S9 (reductive metabolic activation system with Prival modification)

Test concentrations with justification for top dose:

toxicity test: 5000, 1580, 500, 158 and 50.0 µg/plate

Main Assay I ( µg/plate ):
TA1535, TA1537, TA98 −S9: 5000, 2500, 1250, 625, 313
TA1535, TA98 +S9, TA100± S9: 3000, 1500, 750, 375, 188, 93.8
TA1537 +S9: 3000, 1500, 750, 375, 188
WP2 uvrA ± S9: 5000, 2500, 1250S9: 625, 313, 156

Main Assay II ( µg/plate ):
TA1535 −S9, WP2 uvrA, TA98, TA1537 ± S9 : 3000, 1500, 750, 375, 188
TA100 ± S9: 3000, 1500, 750, 375, 188, 93.8
TA1535 + S9: 1500, 750, 375, 188, 93.8, 46.9

Vehicle / solvent:

sterile distilled water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

congo red

methylmethanesulfonate

other: 2-aminoanthracene; Trypan Blue Solution 0.4%

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration : triplicate
- Number of independent experiments : two main assays

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable):
- Test substance added in plate incorporation method for Main Assay I and pre-incubation method for Main Assay II

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable:
- Incubation: 72h at 37°C.After this period of incubation, plates were immediately scored by counting the number of revertant colonies on each plate.


METHODS FOR MEASUREMENT OF CYTOTOXICITY :
thinning of the background lawn

METHODS FOR MEASUREMENTS OF GENOTOXICIY : number of revertants, histidine independent for S. Typhimurium and typtophan indipendent for E. Coli

- OTHER:

Evaluation criteria:

For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at the two highest doses

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:

cytotoxicity

Remarks:

at the highest tested dose

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:

cytotoxicity

Remarks:

at the two highest doses

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:

cytotoxicity

Remarks:

at the highest dose

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:

cytotoxicity

Remarks:

at the highest dose

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No precipitation of the test item, evident to the unaided eye, was observed at the end of the incubation period at any concentration, in any experiment, in the absence or presence of S9 metabolism. In both assays, plates treated with the test item presented a dose dependent blue colour of the agar (yellow-green in the presence of the reductive metabolic system) which did not interfere with the scoring of colonies.

Conclusions:

The substance was tested for gene mutation in bacteria following OECD 471. Under the experimental conditions the test item does not induce reverse mutation in Salmonella typhimurium or Escherichia coli strains in the absence or presence of S9 metabolism (rat liver or Hamster liver/Prival method) in plate incorporation and pre-incubation method.

Executive summary:

The test item was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy. The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbital and 5,6-benzoflavone (standard metabolic activation) in Main Assay I, and liver S9 fraction from uninduced hamsters (reductive metabolic activation system with Prival modification) inMain Assay II. The test item was used as a solution in sterile water for injection. The test item was assayed in the toxicity test at a maximum concentration of 5000 µg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 µg/plate. No precipitation of the test item was observed at the end of the incubation period at any concentration. Moderate toxicity was observed with all tester strains at the highest dose level both in the absence and presence of S9 metabolism.

No relevant increase in revertant numbers was observed with any tester strain at any dose level, in the absence or presence of S9 metabolism.

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

Remarks:

Type of genotoxicity: genome mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Full report; GLP; current guideline method performed on the analogue substance

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS- Source: Harlan Winkelmann Borchen Germany- Age at study initiation: 8 to 12 weeks- Weight at study initiation: 28 to 43 g- Assigned to test groups randomly: yes- Fasting period before study: no data- Housing: males: single; females: groups of

Route of administration:

intraperitoneal

Vehicle:

Vehicle(s)/solvent(s) used: physiol. saline;

Details on exposure:

Dose: preliminary test (toxicity): 100-500 mg/kg bwmain test: 150 mg/kg bwPREPARATION OF DOSING SOLUTIONS:Telon Echtschwarz LD was dissolved in physiological saline solution, using a magnetic stirrer for 30 minutes, stirred with a magnetic mixer during administration and injected intraperitoneally.Administration volume was 10 ml/kg bw

Duration of treatment / exposure:

16, 24, or 48 hours

Frequency of treatment:

single

Remarks:

Doses / Concentrations:150 mg/kg bwBasis:other: nominal injected

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide; - Justification for choice of positive control(s): routinely used positve control- Route of administration: intraperitoneal- Doses / concentrations: 20 mg/kg bw

Tissues and cell types examined:

Bone marrow (femur)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:The selection of the Telon Echtschwarz LD dose was based on a pilot test, in which groups of five animals, including both males and females, were intraperitoneally administered 100 mg/kg, 150 mg/kg, 250 mg/kg and 500 mg/kg Telon Echtschwarz LD. The following symptoms were recorded for up to 48 hours, starting at 100 mg/kg: apathy, black discoloration of hairless parts of skin, staggering gait, sternal recumbency, spasm, difficulty in breathing and eyelids stuck together. In addition, 1 of 5 animals died in the 150 mg/kg group and 5 of 5 animals died in the 250 and 500 mg/kg groups.Based on these results, 150 mg/kg Telon Echtschwarz LD was chosen as MTD for this test.TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):DETAILS OF SLIDE PREPARATION:At least one intact femur was prepared from each sacrificed animal (not pretreated with a spindle inhibitor). A suitable instrument was used to sever the pelvic bones and lower leg. The femur was separated from muscular tissue.The lower-Ieg stump, including the knee and all attached soft parts, was separated in the distal epiphyseal cartilage by a gentle pull at the distal end.The proximal end of the femur was opened at its extreme end with a suitable instrument, e.g. fine scissors, making visible a small opening in the bone-marrow channel. The femur was then completely immersed in the calf serum and pressed against the wall of the tube, to prevent its slipping off.A suitable tube was filled with sufficient fetal calf serum A small amount of serum was drawn from the tube into a suitable syringe with a thin cannula. The cannula was pushed into the open end of the marrow cavity. The femur was then completely immersed in the calf serum and pressed against the wall of the tube, to prevent its slipping off.The contents were then flushed several times and the bone marrow was passed into the serum as a fine suspension.Finally, the flushing might be repeated from the other end, after it had been opened.The tube containing the serum and bone marrow was centrifuged in a suitable centrifuge at approximately 1000 rpm for five minutes. The supernatant was removed with a suitable pipette (e.g. Pasteur pipette), leaving only a small remainder.The sediment was mixed to produce a homogeneous suspension.One drop of the viscou suspension was placed on a well cleaned slide and spread with a suitable object, to allow proper evaluation of the smear.The labeled slides were dried overnight. If fresh smears needed to be stained, they needed to be dried with heat for a short period.The staining of smearsThe smears were stained automatically with an Ames HemaTek Slide Stainer from the Miles Company. The slides were then "destained" with methanol, rinsed with deionized water and left to dry.METHOD OF ANALYSIS:Coded slides were evaluated using a light microscope at a magnification of about 1000. Micronuclei appear as stained chromatin particles in the anucleated erythrocytes. They can be distinguished from artifacts by varying the focus. Normally, 1000 polychromatic erythrocytes were counted per animal. The incidence of cells with micronuclei was established by scanning the slides in a meandering pattern.

Evaluation criteria:

A test was considered positive if, at any of the intervals, there was a relevant and significant increase in the number of polychromatic erythrocytes showing micronuclei in comparison to the negative control.A test was considered negative if there was no relevant or significant increase in the rate of micronucleated polychromatic erythrocytes at any time. A test was also considered negative if there was a significant increase in that rate which, according to the laboratory's experience was within the range of negative controls.In addition, a test was considered equivocal if there was an increase of micronucleated polychromatic erythrocytes above the range of attached historical negative controls, provided the increase was not significant and the result of the negative control was not closely related to the data of the respective treatment group. In this case, a second test had to be performed at the most sensitive interval.An assay was considered acceptable if the figures of negative and positive controls were within the expected range, in accordance with the laboratory's experience and/or the available literature data.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY- Dose range: 100 - 500 mg /kg- Solubility: soluble- Clinical signs of toxicity in test animals: apathy, black discoloration of hairless parts of skin, staggering gait, sternal recumbency, spasm, difficulty in breathing and eyelids stuck together. In addition, 1 of 5 animals died in the 150 mg/kg group and 5 of 5 animals diedin the 250 and 500 mg/kg groups.- Harvest times: 48 hoursRESULTS OF DEFINITIVE STUDY- Induction of micronuclei (for Micronucleus assay): no- Ratio of PCE/NCE (for Micronucleus assay): see table- Appropriateness of dose levels and route: intrapreitoneal administration secured target tissue exposure; the dose showed clear signs of systemic toxicity (mortality) in a preliminary test.- Statistical evaluation:

Results of micronucleus test with Telon Echtschwarz LD after acute peritoneal treatment with 150 mg/kg bw

group	Number of evaluated PCE	NCE/1000 PCE	Micronucelated cells per 1000 NCE	Micronucleated Cells per 1000 PCE
negative control	10000	1056 +/-386	1,2 +/-1,2	1,3 +/-0,7
test 16 hours	10000	2370 +/-1258	1,2 +/-1,0	1,6 +/-1,6
test 24 hours	10000	1537 +/-593	1,1 +/-0,8	2,0 +/-1,7
test 48 hours	10000	1733 +/-1012	0,5 +/-0,7	1,6 +/-0,9
positive control	10000	674 +/-232	0,4 +/-0,8	14,9 +/-9,8

Conclusions:

Interpretation of results (migrated information): negativeThe analogue substance was tested in an vivo micronucleus test following OECD 474. Under the experimental conditions the analogue substance did not cause the formation of micronuclei in mice.

Executive summary:

The analogue substance was examined in mice in vivo for the formation of micronucleated erythrocytes.

Male as well as female mice were treated by intraperitoneal injection with 150 mg/kg bw.

The dose range was 0 (vehicle), 100 to 500 mg/kg bw in a preliminary test and 150 mg/kg bw in the main study. Cyclophosphamide was used as a positive control. Cells were harvested 16, 24 and 40 hours after treatment.

Positive as well as negative controls gave the expected results.

Toxicity was noted at doses of 150 mg/kg bw and above.

No increases of micronucleated cells were observed. The test item is not considered clastogenic in this test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The analogue substance was tested for in vitro mutagenicity (HPRT) with negative results. The trahet substance was tsetd for in vitro bacterial mutagenicity test following OECD 471 that resulted negative.

The negative results of the tests are supported and confirmed in an in vivo micronucleus study in mice using intraperitoneal application, which also produced no indication of genetic damage.

Based on the read across considerations, the test item is not considered to have mutagenic potential

Justification for classification or non-classification

Based on the results of in vitro and in vivo mutagenic tests, no classification for genotoxicity is warranted under Regulation 1272/2008