[8-[(4-amino-3-nitrophenyl)azo]-7-hydroxy-2-naphthyl]trimethylammonium chloride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

This study was performed to investigate the potential of B 007 ( [8-[(4-amino-3-nitrophenyl)azo]-7-hydroxy-2-naphthyl]trimethylammonium chloride) to induce gene mutations according to the plate incorporation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102. The assay was performed with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations of 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate in the main experiment. The plates incubated with the test item showed reduced background growth in nearly all strains used at higher concentrations with and without metabolic activation. Toxic effects, evident as a reduction in the number of revertants, occurred in all strains, except in strain TA 1535 with and without metabolic activation. Substantial and dose dependent increase in revertant colony numbers was observed following treatment with B 007 in strains TA 1537 and TA 98 in the presence and absence of metabolic activation. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did induce gene mutations by frameshifts in the genome of strains TA 1537 and TA 98 in the presence and absence of metabolic activation. The test chemical did not induce gene mutation in the rest of the strains TA98, TA100 and TA102 in the presence and absence of S9 metabolic activation system.

Link to relevant study records

Reference

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

Justification for type of information:

Data is from study report

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

other: Commission Directive 2000/32/EC, L 1362000, Annex 40", dated May 19. 2000.

Principles of method if other than guideline:

The experiments were performed to assess the potential of the test item to induce gene mutations by means of the Salmonella typhimurium reverse mutation assay. Pre-
Experiment and experiment I were performed as a plate incorporation assay. Since a positive result was obtained in this experiment, a second experiment was not required.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Test Item: B 007 ([8-[(4-amino-3-nitrophenyl)azo]-7-hydroxy-2-naphthyl]trimethylammonium chloride)
- Lot/batch No.of test material: 64960101
- Expiration date of the lot/batch: August 15, 2024
- Purity: 95.6 area%

RADIOLABELLING INFORMATION (not applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In the refrigerator (2-8 °C), protected from light
- Stability under test conditions:Not indicated by the sponsor
- Solubility and stability of the test substance in the solvent/vehicle:No data
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:No data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: On the day of the experiment, the test item B 007 was dissolved in deionised water.
- Preliminary purification step (if any): No data
- Final dilution of a dissolved solid, stock liquid or gel: No data
- Final preparation of a solid: No data

OTHER SPECIFICS: No data

Target gene:

The histidine dependent strains are derived from S. typhimurium strain LT2 through a mutation in the histidine locus. Additionally due to the "deep rough" (rfa-) mutation they possess a faulty lipopolysaccharide envelope which enables substances to penetrate the cell wall more easily. A further mutation (deletion of the uvrB gene) causes an inactivation of the excision repair system. The latter alteration also includes a deletion in the nitrate reductase and biotin genes. In the strains TA 98, TA 100, and TA 102 the R-factor plasmid pKM 101 carries umu DC analogous genes that are involved in error-prone repair and the ampicillin resistance marker. The strain TA 102 does not contain the uvrB--mutation and is excision repair proficient. Additionally, TA 102 contains the multicopy plasmid pAQ1 carrying the hisG428 mutation (ochre mutation in the hisG gene ) and a tetracycline resistance gene.

In summary, the mutations of the TA strains used in this study can be described as follows:
Salmonella typhimurium
Strains Genotype Type of mutations indicated
TA 1537 his C 3076; rfa-; uvrB-: frame shift mutations
TA 98 his D 3052; rfa-; uvrB-;R-factor frame shift mutations
TA 1535 his G 46; rfa-; uvrB-: base-pair substitutions
TA 102 his G 428; rfa-; uvrB+;R-factor base-pair substitutions
TA 100 his G 46; rfa-; uvrB-;R-factor base-pair substitutions

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 B. Ames et al. and D.Maron and B. Ames. In this way it was ensured that the experimental conditions set down by Ames were fulfilled.
The bacterial strains TA 1535, TA 1537, TA 98, TA 100, and TA 102 were obtained from Trinova Biochem GmbH (35394 Gießen, Germany).

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

liver microsomal activation

Test concentrations with justification for top dose:

In the pre-experiment the concentration range of the test item was 3 – 5000 μg/plate. The pre-experiment is reported as part of experiment I. Since toxic effects were observed eight concentrations were tested in experiment I and 5000 μg/plate were chosen as maximal concentration.

The concentration range included two logarithmic decades. The following concentrations were tested:

Pre-Experiment / Main Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Vehicle / solvent:

With metabolic activation
Strains: TA 1535, TA 1537, TA 98, TA 100, TA 102 - DMSO

Without metabolic activation
Strains: TA 1535, TA 100 - water deionised
Strains: TA 1537, TA 98 - DMSO
Strain: TA 102 - water deionised

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

Concentration: 10 μg/plate

Positive control substance:

sodium azide

Remarks:

For Strains: TA 1535, TA 100 without metabolic activation.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

Concentration: 10 μg/plate in TA 98, 50 μg/plate in TA 1537

Positive control substance:

other: 4-nitro-o-phenylene-diamine, 4-NOPD

Remarks:

For strains: TA 1537, TA 98 without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

Concentration: 4.0 μL/plate

Positive control substance:

methylmethanesulfonate

Remarks:

For Strain: TA 102 without metabolic activation.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

Concentration: 2.5 μg/plate (10.0 μg/plate in TA 102)

Positive control substance:

other: 2-aminoanthracene, 2-AA

Remarks:

For Strains: TA 1535, TA 1537, TA 98, TA 100, TA 102 with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation)

Storage
The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO (MERCK, D-64293 Darmstadt) in liquid nitrogen.

Precultures
From the thawed ampoules of the strains 0.5 mL bacterial suspension was transferred into 250 mL Erlenmeyer flasks containing 20 mL nutrient medium. A solution of 20 μL ampicillin (25 μg/mL) was added to the strains TA 98, TA 100, and TA 102. Additionally 20 μL tetracycline (2 μg/mL) was added to strain TA 102. This nutrient medium contains per litre:

8 g Merck Nutrient Broth (MERCK, D-64293 Darmstadt)
5 g NaCl (MERCK, D-64293 Darmstadt)

The bacterial cultures were incubated in a shaking water bath for 4 hours at 37° C.

SELECTION AGENT (mutation assays):
Selective Agar
The plates with the minimal agar were obtained from E. Merck, D-64293 Darmstadt.

Overlay Agar
The overlay agar contains per litre:
6.0 g MERCK Agar Agar*
6.0 g NaCl*
10.5 mg L-Histidine x HCl x H2O*
12.2 mg Biotin*
* (MERCK, D-64293 Darmstadt)
Sterilisations were performed at 121° C in an autoclave.

Experimental Performance
For each strain and dose level, including the controls three plates were used.
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 (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),
- 2000 μL Overlay agar
After solidification the plates were incubated upside down for at least 48 hours at 37° C in the dark

Rationale for test conditions:

No data

Evaluation criteria:

The Salmonella typhimurium reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of our historical data
- the positive control substances should produce a significant increase in mutant colony frequencies

Evaluation of Results
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.

A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

The colonies were counted using the Petri Viewer Mk2 (Perceptive Instruments Ltd, Suffolk CB 7BN, UK) with the software program Ames Study Manager. The counter was connected to an IBM AT compatible PC with printer which printed out both, the individual and mean values of the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates. Due to the precipitation of the test item the colonies were partly counted manually.

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100, and TA 102.

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test item B 007 was assessed for its potential to induce gene mutations according to the plate incorporation test using Salmonella typhimurium strains TA 1535, TA 1537,
TA 98, TA 100, and TA 102.The assay was performed with and without liver microsomal activation. Each concentration and the controls, were tested in triplicate. The test item was tested at the following concentrations:
Pre-Experiment / Main Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Substantial and dose dependent increase in revertant colony numbers was observed following treatment with B 007 in strains TA 1537 and TA 98 in the presence and absence
of metabolic activation. The number of colonies reached or exceeded the threshold of twice (strain TA 98) the number of the corresponding solvent control at concentrations as
low as 10 μg/plate (without S9 mix) and 33 μg/plate (with S9 mix) and above and thrice (strain TA 1537) the number of the corresponding solvent control at concentrations 333
μg/plate and above with and without S9 mix, whereas at 2500 and 5000 μg/plate the number of colonies was reduced due to overlapping toxic effects.

Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.

In the pre-experiment, the number of colonies did not quite reach the lower limit of our historical control data in the negative control of strain TA 100 without metabolic activation.
Since this deviation is rather small, this effect is judged to be based upon statistical fluctuations and has no detrimental impact on the outcome of the study.

Remarks on result:

other: The test item did induce gene mutations by frameshifts.

Conclusions:

It can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did induce gene mutations by frameshifts in the genome of strains TA 1537 and TA 98 in the presence and absence of metabolic activation. The test chemical did not induce gene mutation in the rest of the strains TA98, TA100 and TA102 in the presence and absence of S9 metabolic activation system.

Executive summary:

This study was performed to investigate the potential of B 007 ( [8-[(4-amino-3-nitrophenyl)azo]-7-hydroxy-2-naphthyl]trimethylammonium chloride) to induce gene mutations according to the plate incorporation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102.

 

The assay was performed with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

 

Pre-Experiment / Main Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

 

The plates incubated with the test item showed reduced background growth in nearly all strains used at higher concentrations with and without metabolic activation.

 

Toxic effects, evident as a reduction in the number of revertants, occurred in all strains,except in strain TA 1535 with and without metabolic activation.

 

Substantial and dose dependent increase in revertant colony numbers was observed following treatment with B 007 in strains TA 1537 and TA 98 in the presence and absence of metabolic activation.

 

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

  

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did induce gene mutations by frameshifts in the genome of strains TA 1537 and TA 98 in the presence and absence of metabolic activation. The test chemical did not induce gene mutation in the rest of the strains TA98, TA100 and TA102 in the presence and absence of S9 metabolic activation system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Gene mutation in vitro:

Various data available for the target chemical were reviewed to determine the gene mutation ability for the test chemical of [8-[(4-amino-3-nitrophenyl)azo]-7-hydroxy-2-naphthyl] trimethylammonium chloride. The studies are as mentioned below:

 

This study was performed to investigate the potential of B 007 ( [8-[(4-amino-3-nitrophenyl)azo]-7-hydroxy-2-naphthyl]trimethylammonium chloride) to induce gene mutations according to the plate incorporation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102. The assay was performed with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations of 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate in the main experiment. The plates incubated with the test item showed reduced background growth in nearly all strains used at higher concentrations with and without metabolic activation. Toxic effects, evident as a reduction in the number of revertants, occurred in all strains, except in strain TA 1535 with and without metabolic activation. Substantial and dose dependent increase in revertant colony numbers was observed following treatment with B 007 in strains TA 1537 and TA 98 in the presence and absence of metabolic activation. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did induce gene mutations by frameshifts in the genome of strains TA 1537 and TA 98 in the presence and absence of metabolic activation. The test chemical did not induce gene mutation in the rest of the strains TA98, TA100 and TA102 in the presence and absence of S9 metabolic activation system.

 

In vitro Mouse Lymphoma assay (tk locus) was carried out for gene mutations at the tk locus of mouse lymphoma cells both in the absence and presence of S9 metabolic activation. Test concentrations were based on the results of a pre-test on toxicity measuring relative suspension growth. Test chemical conc. used for the two experiment was given below- Experiment I: 8.1, 16.3, 32.5, 65.0 and 97.5 μg/ml without S9-mix, 16.3, 32.5, 65.0, 130.0 and 195.0 μg/ml with S9-mix and Experiment II: 8.0, 16.0, 32.9, 64.0, 128.0 and 192.0 μg/ml without. Deionised water was used as a solvent. In the main test, cells were treated for 4 h or 24 h (without S9 in experiment II only) followed by an expression period of 72 or 48 h to fix the DNA damage into a stable tk mutation. Liver S9 fraction from phenobarbital/β-naphthoflavone- induced rats was used as exogenous metabolic activation system. Toxicity was measured in the main experiments as percentage relative total growth of the treated cultures relative to the total growth of the solvent control cultures. Negative and positive controls were in accordance with the OECD guideline. In experiment I, precipitation was noted at 97.5 and 130.0 μg/ml without S9-mix and at 195.0 and 260.0 μg/ml with S9-mix; in experiment II precipitation occurred at 128.0 and 192.0 μg/ml. The appropriate level of toxicity (10-20% survival after the highest dose) was not reached in experiment with S9-mix pointing to insufficient exposure of the cells. Both in experiment I and II no biological relevant and dose dependent increase in the number mutant colonies was observed independent of the presence or absence of S9-mix. The test substance(8-((4-Amino-3-nitrophenyl)azo)-7-hydroxy-2-naphthyl)trimethylammonium chloride did not induce the mutation in theL5178Y Mouse lymphoma cells(with and without metabolic activation system). Thus, the test was considered to be negative. Thus, it can be concluded that the test substance has negative genetic toxicity effects and based on the CLP criteria for classification, it cannot be classified as genotoxic.

 

In vitro micronucleus test was carried out using Chinese hamster V79 cells. Different conc. of test chemical was used in the various experiment. Deionised water was used as a vehicle. Liver S9 fraction from phenobarbital/β-naphthoflavone-induced rats was used as exogenous metabolic activation system. A pretest on cell growth inhibition (XTT assay) with 4 h treatment was performed in order to determine the toxicity of test substance, the solubility during exposure and thus the test concentrations for the main micronucleus test. On the basis of pre-test (range finding study) and the occurrence of precipitation of test chemical, 4100 μg/ml (≈ 10 mM the prescribed maximum concentration) was chosen as top concentration in experiment IA. To corroborate the data of this experiment in the absence of S9-mix, a confirmatory experiment (experiment IB) was performed with a top dose of 500 μg/ml. Dose selection in experiment IIA was influenced by Basic Brown 17 toxicity and precipitation observed in experiment I. Due to the steep dose toxicity curve, a repeat experiment (experiment IIB) was performed with narrower dilution steps to proof if genotoxicity observed at highly toxic concentrations far below the 40% of control level was an artificial finding. The treatment period in the main test was 4 h in experiment I (without and with S9-mix) and in experiment II (with S9-mix) or 20 h in experiment II (without S9-mix). Harvest time was 24 h or 48 h (experiment II with S9-mix only) after the beginning of culture. For assessment of cytotoxicity a XTT test was additionally carried out in parallel to the main micronucleus test. Under the experimental conditions, the test substance(8-((4-Amino-3-nitrophenyl)azo)-7-hydroxy-2-naphthyl)trimethylammonium chloridedid not induce an increase in micronucleated cells and thus, the test was considered to be negative.Thus, it can be concluded that the test substance has negative genetic toxicity effects and based on the CLP criteria for classification, it cannot be classified as genotoxic.

 

In a study cited in Scientific Committee on Consumer Safety (2014), Mammalian cell gene mutation assay (hprt locus) was carried out using L5178Y Mouse lymphoma cells. A pre-test on toxicity, using identical conditions as in the main test, was performed to determine the concentration range for the mutagenicity experiments using several concentrations between 12.7 and 3256 μg/ml. Test chemical conc. used for the two experiment were Experiment I: 3.1, 6.2, 12.4, 24.8, 49.6 and 99.2 μg/ml without S9-mix, 6.2, 12.4, 24.8, 49.6, 99.2 and 198.4 μg/ml with S9-mix and Experiment II- 3.1, 6.2, 12.4, 24.8, 37.2 and 49.6 μg/ml without S9-mix, 3.1, 6.2, 12.4, 24.8, 49.6 and 99.2 μg/ml with S9-mix. Deionised water was used as a solvent. In the main tests, cells were treated for 4h without and with S9-mix or for 24h without S9-mix followed by an expression period of 48h to fix the DNA damage into stable hprt mutations. A serum concentration of 3% was used. Toxicity was measured as percentage survival of the treated cultures relative to the survival of the solvent control cultures. Negative and positive controls were in accordance with the OECD guideline. In both experiments without and with metabolic activation, precipitation was observed at 49.6 μg/ml and above. Exclusively, in experiment I without metabolic activation, the required relative survival of 10-20% as compared to the untreated control was reached. A biological relevant increase of the mutant frequency was not observed in both experiments with and without metabolic activation. An isolated increase exceeding the limit of three times the mutant frequency of the untreated control was observed in one culture of experiment II with metabolic activation at the highest concentration (99.2 μg/ml). However, precipitation occurred at this concentration and the increase was not concentration dependent. Therefore, this isolated effect was considered not biologically relevant. The test substance(8-((4-Amino-3-nitrophenyl)azo)-7-hydroxy-2-naphthyl)trimethylammonium chloride did not induce the mutation at the hprt locus in theL5178Y Mouse lymphoma cells(with and without metabolic activation system). Thus, the test was considered to be negative. Thus, it can be concluded that the test substance has negative genetic toxicity effects and based on the CLP criteria for classification, it cannot be classified as genotoxic.

Based on the data available for the target chemical, [8-[(4-amino-3-nitrophenyl)azo]-7-hydroxy-2-naphthyl] trimethylammonium chloride is shown to be positive for gene mutation by one study bu the rest studies suggest the chemical to be negative in nature. Hence, the chemical does not exhibit gene mutation in vitro. Hence, the test chemical is not likely to classify as a gene mutant in vitro.

Justification for classification or non-classification

Based on the data available for the target chemical, [8-[(4-amino-3-nitrophenyl)azo]-7-hydroxy-2-naphthyl] trimethylammonium chloride is shown to be positive for gene mutation by one study bu the rest studies suggest the chemical to be negative in nature. Hence, the chemical does not exhibit gene mutation in vitro. Hence, the test chemical is not likely to classify as a gene mutant in vitro.