1-Naphthalenesulfonic acid, 3-diazo-3,4-dihydro-4-oxo-, ester with phenyl(2,3,4-trihydroxyphenyl)methanone 6-diazo-5,6-dihydro-5-oxo-1-naphthalenesulfonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assay: WoE approch, result: positive

 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

April 16-25, 1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

only one strain tested (to reproduce result from previous study), documentation limited

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983

Deviations:

yes

Remarks:

only one strain was tested

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

his-operon

Species / strain / cell type:

S. typhimurium TA 1537

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9-mix, Aroclor 1254 induced

Test concentrations with justification for top dose:

0, 4, 20, 100, 500, 2500 and 5000 µg/plate (1st experiment)
0, 20, 100, 500, 2500, 3750 and 5000 µg/plate (2nd experiment)

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

not specified

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

a dose-related increase in the number of revertants was observed, but this was only exceeding the critical factor of 2 in the second experiment.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

The test substance is mutagenic in the bacterial reverse mutation assay.

Executive summary:

The test item was tested for its mutagenic potential in bacterial cells according to OECD 471 with one Salmonella typhimirium strain (TA 1537). The following doses were tested:

0, 4, 20, 100, 500, 2500 and 5000 µg/plate (1st experiment)

0, 20, 100, 500, 2500, 3750 and 5000 µg/plate (2nd experiment)

the presence of a metabolic activation system a dose-dependent and reproducible increase in the number of revertants was observed with the tester strain TA 1537. In the absence of metabolic activation a dose-related increase in the number of revertants was also observed, but this was only exceeding the critical factor of 2 in the second experiment. However, based on the result of the study, test item is considered to be mutagenic in bacterial cells.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

only 4 bacterial strains tested

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983

Deviations:

no

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9-mix, Aroclor 1254 induced

Test concentrations with justification for top dose:

6 doses from 4 to 5000 µg/plate

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

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) (range-finding study (Experiment 1) and main experiment (Experiment 2))

DURATION
- Exposure duration: 48 - 72 h

NUMBER OF REPLICATIONS: triplicates

DETERMINATION OF CYTOTOXICITY
- Method: revertant colony number and microscopical inspection of the bacterial background lawn

The results of both experiments (exp.1 and 2) were used for determining the mutagenic properties of the test substance.

Evaluation criteria:

A chemical is considered to have a positive response, if it
-produces at least a 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn
-induces a dose-related increase in the mean number of revertants per plate
The results must be reproducible.

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

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

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

In this assay the test substance was considered to be mutagenic.

Executive summary:

The test item was tested for its mutagenic potential in bacterial cells according to OECD 471 with four Salmonella typhimurium strains (TA 100, TA 1535, TA 1537, TA 98). Six test concentration from 4 – 5000 µg/plate were used. In the presence of a metabolic activation system a dose-dependent and reproducible increase in the number of revertants was observed with the tester strain TA 1537. In the absence of metabolic activation a slight increase was also observed, but only in one strain. Therefore the test item was considered to be mutagenic in bacterial cells.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 5 to 7, 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

only 4 strains tested

GLP compliance:

yes

Remarks:

KoreaGLP

Type of assay:

bacterial reverse mutation assay

Target gene:

his-operon

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

Test concentrations with justification for top dose:

experiment I: 0, 8, 40, 200, 1000, 5000 µg/plate
experiment II: 6.9,20.6, 61.7, 185.2, 555.6, 1666.7 µg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

furylfuramide

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) (range-finding study (Experiment 1) and main experiment (Experiment 2))

DURATION
- Exposure duration: 48 - 72 h

NUMBER OF REPLICATIONS: triplicates (experiment I), duplicate (experiment I)

DETERMINATION OF CYTOTOXICITY
- Method: revertant colony number and microscopical inspection of the bacterial background lawn

The results of both experiments (exp.1 and 2) were used for determining the mutagenic properties of the test substance.

Evaluation criteria:

Results are judged as positive if the number of colony increases with dose-response and reaches 2 times that of the control, or if there is statistically significant increase in the number of colonies in any of the tester strains.

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

In the presence of S9-mix, there was significant increase in the number of revertants in strain TA 1537, which reached up to 8 times that of the control and was reproducible.

Conclusions:

The test item was mutagenic in the bacterial reverse mutation assay.

Executive summary:

The test item was tested for its mutagenic potential in bacterial cells according to OECD 471 with four Salmonella typhimurium strains (TA 100, TA 1535, TA 1537, TA 98). The following doses were tested:

experiment I: 0, 8, 40, 200, 1000, 5000 µg/plate

experiment II: 6.9,20.6, 61.7, 185.2, 555.6, 1666.7 µg/plate

In the presence of S9-mix, there was significant increase in the number of revertants in strain TA 1537, which reached up to 8 times that of the control and was reproducible. Therefore the test item was considered to be mutagenic in bacterial cells.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

The test substance was not clastogenic in the in vivo mammalian erythrocyte micronucleus test (reference 7.6.2-1).

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:

July 1st, 1996 to July 18th, 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

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:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1983

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

other: SHOE:NMRI

Details on species / strain selection:

The mouse is the recommended test species for this assay.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Tierzucht Schönwalde GmbH i.G., Schönwalde, Germany
- Age at study initiation: appr. 8 weeks
- Weight at study initiation: males mean: 36.8 g, females mean: 29.8 g
- Housing: five animals per cage
- Diet: ad libitum rat/mice diet ssniff (ssniff GmbH, Soest, Germany)
- Water: tap water ad libitum
- Acclimation period: 1 week

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

Route of administration:

oral: gavage

Vehicle:

- Vehicle/solvent used: sesame oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment, the test item was suspended in sesame oil. A magnetic stirrer was used to keep the preparation homogenous until administration.

Duration of treatment / exposure:

one treatment

Frequency of treatment:

one oral gavage treatment

Post exposure period:

Animals were killed 12, 24 or 48 h after dosing.

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

killing times: 12, 24 or 48 h, 5 males and 5 females each

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

killing times: 12, 24 or 48 h, 5 males and 5 females each

No. of animals per sex per dose:

5 per killing time

Control animals:

yes

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: oral (gavage)
- Doses / concentrations: 50 mg/kg bw

Tissues and cell types examined:

erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
prelinimary toxicity study

DETAILS OF SLIDE PREPARATION:
A suspension of bone marow (taken from the femorae) and fetal bovine serum was formed and centrifuged for 5 minutes at app. 1200 rpm. The supernatant was discarded. The sediment was smeared on a clean slide. Staining was performed with Giemsa solution.

METHOD OF ANALYSIS:
1000 polychromatic erythrocytes were counted for each animal. The number of cells with micronuclei was recorded.

Evaluation criteria:

A substance is considered positive if there is a significant increase in the numbe rof micronucleated polychromatic erythrocytes for at least one of the time points compared with the concurent negative control group.

Statistics:

Wilcoxon-test (one-sided)

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:

No mortality and no clinical signs of toxicity were observed. No macroscopic findings were recorded at dissection.

Please refer to the attached background material.

Conclusions:

The test item was not clastogenic in vivo under the conditions of the present study.

Executive summary:

The test substance was tested in an in vivo micronucleus test according to OECD Guideline 474. The test item was suspended in sesame oil and administered once via gavage at doses of 0 and 2000 mg/kg bw to male and female NMRI mice. The animals were killed either after 12, 24 or 48 h (5/sex/killing time). Endoxan (cyclophosphamide) was used as positive control at a dose of 50 mg/kg bw. The number of polychromatic and normochromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic/normochromatic erythrocytes in both male and female animals remained unaffected by the treatment with the test item and was statistically not different from the control values. The positive control induced a marked statistically significant increase in the number of polychromatic cells with micronuclei. The ratio of polychromatic erythrocytes to normocytes was not changes to a significant extent. Under the conditions of the study, th test item is not mutagenic in this test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Ames test, reference 7.6.1-1

The test item was tested for its mutagenic potential in bacterial cells according to OECD 471 with four Salmonella typhimurium strains (TA 100, TA 1535, TA 1537, TA 98). Six test concentration from 4 – 5000 µg/plate were used. In the presence of a metabolic activation system a dose-dependent and reproducible increase in the number of revertants was observed with the tester strain TA 1537. In the absence of metabolic activation a slight increase was also observed, but only in one strain. Therefore the test item was considered to be mutagenic in bacterial cells.

 

Ames test, reference 7.6.1-2

The test item was tested for its mutagenic potential in bacterial cells according to OECD 471 with one Salmonella typhimirium strain (TA 1537). The following doses were tested: 0, 4, 20, 100, 500, 2500 and 5000 µg/plate (1st experiment); 0, 20, 100, 500, 2500, 3750 and 5000 µg/plate (2nd experiment), the presence of a metabolic activation system a dose-dependent and reproducible increase in the number of revertants was observed with the tester strain TA 1537. In the absence of metabolic activation a dose-related increase in the number of revertants was also observed, but this was only exceeding the critical factor of 2 in the second experiment. However, based on the result of the study, test item is considered to be mutagenic in bacterial cells.

 

Ames test, reference 7.6.1-3

The test item was tested for its mutagenic potential in bacterial cells according to OECD 471 with four Salmonella typhimurium strains (TA 100, TA 1535, TA 1537, TA 98). The following doses were tested: experiment I: 0, 8, 40, 200, 1000, 5000 µg/plate; experiment II: 6.9,20.6, 61.7, 185.2, 555.6, 1666.7 µg/plate. In the presence of S9-mix, there was significant increase in the number of revertants in strain TA 1537, which reached up to 8 times that of the control and was reproducible. Therefore the test item was considered to be mutagenic in bacterial cells.

 

In vivo-study: Micronucleus assay, reference 7.6.2-1

The test substance was tested in an in vivo micronucleus test according to OECD Guideline 474. The test item was suspended in sesame oil and administered once via gavage at doses of 0 and 2000 mg/kg bw to male and female NMRI mice. The animals were killed either after 12, 24 or 48 h (5/sex/killing time). Endoxan (cyclophosphamide) was used as positive control at a dose of 50 mg/kg bw. The number of polychromatic and normochromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic/normochromatic erythrocytes in both male and female animals remained unaffected by the treatment with the test item and was statistically not different from the control values. The positive control induced a marked statistically significant increase in the number of polychromatic cells with micronuclei. The ratio of polychromatic erythrocytes to normocytes was not changes to a significant extent. Under the conditions of the study, the test item is not mutagenic in this test.

Based on these results and additionally taking into account a structurally similar substance (1-Naphthalenesulfonic acid, 6-diazo-5,6-dihydro-5-oxo-, 4-benzoyl-1,2,3-benzenetriyl ester, CAS 5610-94-6), a genotoxic potential can be excluded. This substance was tested according to OECD guidelines 471, 473 and 476, and no clastogenic or mutagenic potential was revealed.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genetic toxicity, the test item is not considered to be classified for genetic toxicity according to Regulation (EC) No 1272/2008 (CLP), as amended for the seventeenth time in Regulation (EU) 2021/849.