Sodium 6-amino-5-[[2-[(cyclohexylmethylamino)sulphonyl]phenyl]azo]-4-hydroxynaphthalene-2-sulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Sodium 6-amino-5-[[2-[(cyclohexylmethylamino)sulphonyl]phenyl]azo]-4-hydroxynaphthalene-2-sulphonate was positive in the Ames test but negative in the in vitro mammalian gene mutation test.

Link to relevant study records

Reference

Reference 1

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:

1997

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:

yes

Remarks:

(only four strains were tested)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Name: FAT 20'003/I
Batch No.: not indicated by the sponsor
Vers. No.: 9-5-97
Aggregate State at Room Temperature: Solid
Colour: red
Purity: approx 90%
Stability in Solvent: Stable for atleast 24 hours at room temperature and under test conditions at 37°C in water, PEG 400, saline, FCA/NaCl-solution and CMC solution.
Storage: room tempearture
Expiration Date: Feb 2001.

On the day of the experiment, the test article FAT 20003/I was dissolved in DMSO. The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Target gene:

Histidine gene

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

The strains are derived from S. typhimurium strain LT2 and due to a mutation in the histidine locus are histidine dependent. Additionally due to the "deep rough" (rfa-minus) mutation they possess a faulty lipopolysaccharide envelope which enables substances to penetrate the cell wall more easily. A further mutation causes a reduction in the activity of an excision repair system. The latter alteration includes mutational processes in the nitrate reductase and biotin genes produced in a UV-sensitive area of the gene named "uvrB-minus". In the strains TA 98, TA 100 and TA 102 the R-factor plasmid pKM 101 carries the ampicillin resistance marker. The strain TA 102 does not contain the uvrB"-mutation. In the strains TA 98 and TA 100 the R-factor plasmid pKM 101 carries the ampicillin resistance marker. Regular checking of the properties of the strains regarding the membrane permeability, ampicillin- and tetracycline-resistance as well as spontaneous mutation rates is performed in the laboratory of RCC Cytotest Cell Research according to Ames et al. In this way it was ensured that the experimental conditions set down by Ames were fulfilled.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

33, 100, 333, 1000, 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Untreated negative controls:

yes

Remarks:

(concurrent untreated)

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

(for TA 1535 and TA100 without metabolic activation)

Untreated negative controls:

yes

Remarks:

(concurrent untreated)

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine

Remarks:

(for TA 1537 and TA98 without metabolic activation)

Untreated negative controls:

yes

Remarks:

(concurrent untreated)

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

(for all strains with metabolic activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation test (experiment I) and the pre-incubation test (experiment II)

For each strain and dose level, including the controls, a minimum of three plates were used.
Experiment 1: The following materials were mixed in a test tube and poured onto the selective agar plates:
- 100 µL: Test solution at each dose level, solvent control, negative control, or reference mutagen solution (positive control),
- 500 µL: S9 mix (for test with metabolic activation) or S9 mix substitution-buffer (for test without metabolic activation),
- 100 µL: Bacteria suspension (cf. test system, pre-culture of the strains),
- 2,000 µL: Overlay agar

Experiment 2: In the pre-incubation assay 100 µL test solution, 500 µL S9 mix /S9 mix substitution buffer and 100 µL bacteria suspension were mixed in a test tube and incubated at 37 °C for 60 minutes. After pre-incubation 2 mL overlay agar (45 °C) was added to each tube. The mixture was poured on minimal agar plates. After solidification the plates were incubated upside down for at least 48 h at 37 °C in the dark.

Evaluation criteria:

The generally accepted conditions for the evaluation of the results are: corresponding background growth on both negative control and test plates as well as normal range of spontaneous reversion rates.
A test substance is considered as positive if either a dose related or reproducible increase in the number of revertants or a significant and reproducible increase for at least one test concentration is induced.
A test substance producing neither a dose related and reproducible increase in the number of revertants nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.
A biologically relevant response is described as follows:
A test substance is considered mutagenic if the number of reversions is at least twice the spontaneous reversion rate in strains TA 98 and TA 100 or thrice on TA 1535 and TA 1537. Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test substance regardless whether the highest dose induced the criteria described above or not.

Statistics:

A statistical analysis of the data is not required.

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:

cytotoxicity

Remarks:

(only in plate incorporation method)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The plates incubated with the test substance showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used. The frequency of spontaneous revertant colony formation in strain TA 1535 in the second experiment slightly exceeded the historical control range (36 colonies versus 10 - 29 colonies) in the absence of metabolic activation. This increase is most likely based upon a slight increase in the bacterial cell density during pre-incubation and does not represent statistical fluctuations since all test points are at or somewhat above the upper limit of the historical range of negative controls. A slight or moderate increase in the bacterial cell density is not uncommon during pre-incubation since small amounts of histidine containing medium is transferred together with the bacteria and the overall dilution during the preincubation step is considerably lower as compared to the direct plate incorporation method. Therefore, this slightly elevated frequency of spontaneous revertants was judged as biologically irrelevant within the scope of the assay.
No substantial increase in revertant colony numbers of any of the four tester strains was observed following treatment with the test substance at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Conclusions:

The test substance was considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Executive summary:

An in vitro study was performed to investigate the potential of the test substance (at ca. 90 % purity) to induce gene mutations according to OECD Guideline 471 in compliance with GLP with deviation (i.e., only four strains were tested).

The assay was performed in two independent experiments both with and without liver microsomal activation using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100. Experiment I was performed as a plate incorporation assay and experiment II was performed as a pre-incubation assay. Each concentration, including the controls, was tested in triplicate. The substance was tested up to 5000 µg/plate.

Slight toxic effects, evident as a reduction in the number of revertants, occurred in strain TA 98 with and without S9 mix at 5000 µg/plate in Experiment I. In Experiment II, no toxic effects were observed. The plates incubated with the test substance showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used. No substantial increase in revertant colony numbers of any of the four tester strains was observed at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Taking into consideration the above findings, the test substance was considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Reference 2

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:

2014

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:

yes

Remarks:

In experiment II without metabolic activation, at a concentration of 200 μg/mL only four petri dishes have been evaluated due to contamination of one petri dish.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

In experiment II without metabolic activation, at a concentration of 200 μg/mL only four petri dishes have been evaluated due to contamination of one petri dish.

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

yes

Remarks:

In experiment II without metabolic activation, at a concentration of 200 μg/mL only four petri dishes have been evaluated due to contamination of one petri dish.

GLP compliance:

yes (incl. QA statement)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

Name: FAT 20003/K TE
Batch No.: AT-0026132600
CAS No.: 32846-21-2
Physical State / Colour: solid / brown
Storage Conditions: room temperature, protected from light
Molecular Weight of Base Form: 517.60 g/mol
Molecular Weight of Salt Form: 540.59 g/mol
Active Components: > 90%
Analysation Date: 07 January 2014
Expiry Date: 20 December 2018

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 Wistar Phenobarbital and ß-Naphthoflavone-induced rat liver S9 mix

Test concentrations with justification for top dose:

Pre-experiment for experiment I (with and without metabolic activation):
2.5, 5, 10, 25, 50, 100, 200, 300, 400, 500, 600 and 750 µg/mL

Pre-experiment for experiment II (only without metabolic activation, 20 h long-term exposure assay):
1.0, 2.5, 5, 10, 25, 50, 75, 100, 125, 150, 175, 200 µg/mL

Experiment I
without metabolic activation: 10, 25, 50, 75, 100, 110, 120 and 130 µg/mL
With metabolic activation: 100, 200, 300, 350, 400, 450, 475, 500 and 525 µg/mL

Experiment II
without metabolic activation: 100, 200, 250, 300, 350, 400, 450, 500, 550 and 600 µg/mL
With metabolic activation: 320, 360, 400, 440, 480, 510, 520 and 535 µg/mL

Vehicle / solvent:

Vehicle (Solvent) used: DMSO
Justificaiton for choice of solvent : The solvent was compatible with the survival of the cells and the S9 activity. The pH-value detected with the test item was within the physiological range.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation; 300 µg/mL

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation; 0.8 and 1.0 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: dissolved in DMSO
DURATION: 4 h (short-term exposure), 20 h (long-term exposure)
Expression time (cells in growth medium): 5 days
Selection time (if incubation with selection agent): about one week

SELECTION 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 evaluated
NUMBER OF CELLS EVALUATED: 400000 cells per flask
DETERMINATION 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.

Key result

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: ≥ 100 μg/mL; experiment I with S9: ≥ 475 μg/mL; Experiment II without S9: ≥ 250 μg/mL; Experiment II with S9:≥ 480 μg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Conclusions:

FAT 20003/K TE is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.

Executive summary:

An experiment was performed to assess the mutagenic potential of FAT 20003/K TE in cell mutation assay (HRPT Locus V79 cells) according to OECD test guideline 476.

 

In a mammalian cell gene mutation assay (HPRT locus, V79 cells cultured in-vitro were exposed to FAT 20003/K TE at following concentrations;

- 10, 25, 50, 75, 100, 110, 120 and 130 µg/mL (without metabolic activation, Experiment I)

- 100, 200, 300, 350, 400, 450, 475, 500 and 525 µg/mL (with metabolic activation, Experiment I)

- 100, 200, 250, 300, 350, 400, 450, 500, 550 and 600 µg/mL (without metabolic activation, Experiment II)

- 320, 360, 400, 440, 480, 510, 520 and 535 µg/mL (with metabolic activation, Experiment II).

FAT 20003/K TE 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 12.0 % for the highest concentration (130 µg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 525 µg/mL with a relative growth of 17.9 %.

 In experiment II without metabolic activation the relative growth was 12.6 % for the highest concentration (600 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 535 µg/mL with a relative growth of 17.3 %.

In experiment I without metabolic activation the highest mutation rate (compared to the solvent control values) of 2.42 was found at a concentration of 110 µg/mL with a relative growth of 31.4 %.

In experiment I with metabolic activation the highest mutation rate (compared to the solvent control values) of 1.43 was found at a concentration of 400 µg/mL with a relative growth of 72.8 %.

In experiment II without metabolic activation the highest mutation rate (compared to the solvent control values) of 0.92 was found at a concentration of 400 µg/mL with a relative growth of 39.3 %.

In experiment II with metabolic activation the highest mutation rate (compared to the solvent control values) of 2.37 was found at a concentration of 480 µg/mL with a relative growth of 58.7 %.

 

In both experiments no biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation). No dose-response relationship was observed.

DMBA and EMS were used as positive controls and showed distinct and biologically relevant effects in mutation frequency.

 

Under the experimental conditions reported, the test item FAT 20003/K TE is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The read-across substance was negative for induction of chromosome damage in an oral mouse micronucleus study.

Additional information

Sodium 6-amino-5-[[2-[(cyclohexylmethylamino)sulphonyl]phenyl]azo]-4-hydroxynaphthalene-2-sulphonate was evaluated for genotoxic potential in the standard Ames/Salmonella gene mutation test and in the in vitro mammalian cell gene mutation test (HPRT-locus). Additionally a structural analogue that was selected as a read-across candidate was evaluated in an in vitro cytogenecity chromosome aberration study as well as in an in vivo chromosome aberration test. Sodium 6-amino-5-[[2-[(cyclohexylmethylamino)sulphonyl]phenyl]azo]-4-hydroxynaphthalene-2-sulphonate was positive in the Ames test but negative in the in vitro mammalian gene mutation test. The read-across candidate was positive in the in vitro mammalian chromosome aberration test. However, the read-across substance was negative for induction of chromosome damage in an oral mouse micronucleus study.

Short description of key information:
Sodium 6-amino-5-[[2-[(cyclohexylmethylamino)sulphonyl]phenyl]azo]-4-hydroxynaphthalene-2-sulphonate was not mutagenic in vitro (Ames test and mammalian cytogenicity). A structural analogue was positive in vitro in mammalian cells for clastogenicity, but did not show evidence of clastogenicity in an in vivo micronucleus test. Hence sodium 6-amino-5-[[2-[(cyclohexylmethylamino)sulphonyl]phenyl]azo]-4-hydroxynaphthalene-2-sulphonate is considered to be neither mutagenic nor clastogenic, and hence not genotoxic.

Justification for classification or non-classification

Based on the above stated assessment of the genotoxic potential of Sodium 6-amino-5-[[2-[(cyclohexylmethylamino)sulphonyl]phenyl]azo]-4-hydroxynaphthalene-2-sulphonate (Ames test negative, in vitro mammalian cell gene mutation test negative; in vitro mammalian chromosome aberration test and negative and in vivo bone marrow chromosome aberration test in the structural analogue) the substance is deemed non-genotoxic and accordingly does not need to be classified according to Council Directive 2001/59/EC (28th ATP of Directive 67/548/EEC) and according to CLP (Regulation (EC) No 1272/2008 Of The European Parliament And Of The Council) as implementation of UN-GHS in the EU.