Disodium [N-[7-hydroxy-8-[(2-hydroxy-5-mesylphenyl)azo]-1-naphthyl]acetamidato(2-)][2-hydroxy-3-[(2-hydroxy-1-naphthyl)azo]-5-nitrobenzenesulphonato(3-)]chromate(2-)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity, in vitro

The study was performed to investigate the potential of the Similar substance 01 to induce gene mutations according to the plate incorporation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100. The assay was performed in two independent experiments both with and without liver microsomal activation. It was concluded that the metabolites of the substance exerted a mutagenic action on strain S. typhimurium TA 98.

The Similar substance 03 was assessed for its mutagenic potential in the chromosome aberration test in human lymphocytes in vitro in the absence and presence of S9 mix (supporting study). In the described study and under the experimental conditions reported, the test article (read across substance did not induce structural chromosome aberrations in human lymphocytes in vitro.

In the supporting pre-experiment on toxicity (scoring the mitotic index) with and without S9 mix treatment with 500 µg/ml reduced the mitotic index to about 50 % as compared to the negative controls. The Similar substance 03 did not relevantly increase the number of cells carrying structural chromosome aberrations neither in the absence nor in the presence of S9 mix.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

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

Adequacy of study:

key study

Study period:

1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Justification for type of information:

Justification for read across is detailed in the report attached to the IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only four tester strains tested; no tester strain to detect cross-linking mutagens was included

Principles of method if other than guideline:

None

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

None

Target gene:

Histidine for Salmonella

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Strain Type of MutationS. typhimurium TA 100 base-pair substitutionS. typhimurium TA 1535 base-pair substitutionS. typhimurium TA 98 frame-shiftS. typhimurium TA 1537 frame-shift

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat-liver post mitochondrial supernatant (S9 fraction)

Test concentrations with justification for top dose:

0.2 to 2000 µg

Vehicle / solvent:

bidistilled water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without S9: strains TA 1535 and TA 100 - MNNG, for Strain TA 98 - Daunomycine and for strain TA 1537 - 9-aminoacridine; with S9: 2-anthramine was used for all the strains

Details on test system and experimental conditions:

INDUCTION OF RAT LIVER ENZYMESThree male Sprague-Dawley rats weighing approximately 200 grams were given an i.p. injection of Aroclor 1254 (Monsanto) in peanut oil at a dose of 500 mg/kg.Five days after the injection, the rats were anaesthetized with ether, decapitated and the livers removed in a sterile manner. The livers were homogenizedin twice their volume of sterile 0.15 M KCl, pooled together and centrifuged for 15 min at 9000 xg (Beekman refrigerated ultracentrifuge). The supernatant(termed the S-9 fraction) was aliquoted into sterile tubes and frozen at -70°C.TEST SUBSTANCE AND CONCENTRATIONSA sample of the product FAT 20013/B (a brown dyestuff) was tested in S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 at five dose levels: 0.2, 2, 20, 200 and 2000 (jg (or nl) per Petri dish. The test substance was dissolved in distilled water and every concentration was tested in triplicate.

Rationale for test conditions:

None

Evaluation criteria:

The criteria of mutagenicity used in this test are a doubling of the spontaneous reversion rate and a dose-effect relationship.

Statistics:

None

Key result

Species / strain:

other: For strains TA 1535, TA 1537 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

None

None

Conclusions:

It is concluded that the metabolites exerted a mutagenic action on strain S. typhimurium TA 98.

Executive summary:

The study was performed to investigate the potential of Fteh Similar substance 01 to induce gene mutations according to the plate incorporation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100. The assay was performed in two independent experiments both with and without liver microsomal activation.

Under the experimental conditions defined in the protocol, and employing a doubling of the spontaneous reversion rate and dose-effect relationship as criteria of mutagenicity, the product was found mutagenic for S. typhimurium strain TA 98. No mutagenic effect was observed with the strains TA 1535, TA 1537 and TA 100. With TA 98, the evidence of mutagenicity existed at 2000 µg in the presence of S-9 mix, and at 200 µg and 2000 µg of product per Petri dish without S-9 mix. In the absence of S-9 mix, at 2000 µg of product per plate, a toxic effect was noted with a dispersed background lawn. A complete disappearance of the background lawn was observed with the strains TA 1535 and TA 1537 at 2000 µg in the absence of S-9 mix.

It is concluded that the metabolites of the substance exerted a mutagenic action on strain S. typhimurium TA 98.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Genetic toxicity, in vivo

An in vivo study was performed to investigate the potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. None of the animals expressed toxic reactions. In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, the Similar substance 01 is considered to be non-mutagenic in this micronucleus assay.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

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

Adequacy of study:

key study

Study period:

1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Justification for read across is detailed in the report attached to the IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Principles of method if other than guideline:

None

GLP compliance:

yes

Type of assay:

mammalian germ cell cytogenetic assay

Specific details on test material used for the study:

None

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

None

Sex:

male/female

Details on test animals or test system and environmental conditions:

Strain: NMRISource: BRL, CH-4414 FüllinsdorfNumber of Animals: 72 (36 males/36 females)Initial Age at Start of Acclimatization: 8-12 weeksAcclimatization: minimum 5 daysInitial Body Weight at Start of Treatment: males mean value 31.5 g (SD ± 3.2 g)females mean value 25.1 g (SD ± 1.8 g)

Route of administration:

oral: gavage

Vehicle:

Vehicle: Deionised water

Details on exposure:

It is generally recommended to use the maximum tolerated dose or the highest dose that canbe formulated and administered reproducibly or 2000 mg/kg as the upper limit for non-toxictest articles.The maximum tolerated dose level is determined to be the dose that causes toxic reactionswithout having major effects on survival within 48 hours.The volume to be administered should be compatible with physiological space available.Three adequate spaced dose levels extending over a single log range were applied at thecentral sampling interval 24 h after treatment. For the highest dose level an additionalsample was taken at 48 h after treatment.

Duration of treatment / exposure:

48 hours

Frequency of treatment:

once

Post exposure period:

None

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

6 animals per sex per dose

Control animals:

yes

Positive control(s):

Cyclophosphamide; - Justification for choice of positive control(s): recommended by the guidelines - Route of administration: oral - Doses : 10 mg/kg b.w.- Frequency: once

Tissues and cell types examined:

The animals were sacrificed by cervical dislocation: The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Griinwald (MERCK, D-64293 Darmstadt)/Giemsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mounted with EUKITT (KINDLER, D-79110 Freiburg). At least one slide was made from each bone marrow sample.

Details of tissue and slide preparation:

Analysis of Cells:Evaluation of the slides was performed using NIKON microscopes with lOOx oil immersion objectives. At least 1000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in normochromatic erythrocytes per 1000 the PCEs. The analysis was performed with coded slides.Five animals per sex and group were evaluated as described. The remaining 6th animal of each test group was evaluated in case an animal had died in its test group.

Evaluation criteria:

The study is considered valid if the following criteria are met:- the vehicle controls are in the range of our historical control data (0.03 - 0.26 % PCEs with micronuclei).- the positive controls show substantially increased values- more than 80 % of animals are évaluable

Statistics:

None

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

None

None

Conclusions:

non-mutagenic in in vivo micronucleus assay.

Executive summary:

An in vivo study was performed to investigate the potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test article was formulated in deionised water . Deionised water was used as vehicle control. The volume administered orally was 20 ml/kg b.w.. 24 h and 48 h after a single administration of the test article the bone marrow cells were collected for micronuclei analysis. Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. 1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test article the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 1000 PCE. The following dose levels of the test article were investigated: 24 h preparation interval: 200, 670 and 2000 mg/kg b.w.. 48 h preparation interval: 2000 mg/kg b.w.. The highest guideline-recommended dose (2000 mg/kg) was estimated by a pre-experiment to be suitable. None of the animals expressed toxic reactions.

After treatment with the highest dose of test article the number of NCEs was increased as compared to the corresponding vehicle controls thus indicating that the Similar substance 01 had cytotoxic effectiveness in the bone marrow. In comparison to the corresponding vehicle controls there was no significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test article and with any dose level used. 40 mg/kg b.w. cyclophosphamide administered per os was used as positive control which showed a statistically significant increase of induced micronucleus frequency.

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, the Similar substance 01 is considered to be non-mutagenic in this micronucleus assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

 Based on negative results in in vitro chromosomal aberration assay and mammalian cell mutation assay (HPRT) with the read across substance 03 as well as the in vivo micronucleus assay with Similar substance 03, the registered substance can be considered to be non-genotoxic. Hence, the substance is not classified.