N-(4-amino-9,10-dihydro-3-methoxy-9,10-dioxo-1-anthryl)benzenesulphonamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Disperse Red 86:1 was found to have mutagenic action in the bacterial reverse mutation assays, however, the source chemical, Disperse Red 86, could not lead to mutagenic action in an in vitro mammalian cell gene mutation assay.

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:

2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted July 21, 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Directive 2000!32/EC, L 1362000. Anllex 4D", dated May 19, 2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Guidelines: "Kanpoan No. 287 -- Envlrorlment Protection Agency" Eisei No. 127 -- Ministry 01 Health & Welfare" "Heisei 09/10131 Kikyoku No. 2 -- Ministry of In!erna!ional Trade & Industry"

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 09979EB5
- Expiration date of the lot/batch: March 14, 2011

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature

Target gene:

Salmonella typhimurium histidine (his) and the E. coli tryptophan (trp)

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:

S9 mixType and composition of metabolic activation system:
- source of S9 : rat liver
- method of preparation of S9 mix:
The S9 is prepared from 8 - 12 weeks old male Wistar Hanlbm rats, weight approx, 220 - 320 g induced by applications of 80 mglkg b.w. Phenobarbital Lp (Desitin; D-22335 Hamburg) and ß-Naphthoflavone p,o. (Aldrich, D-89555 Steinheim) each on three consecutive days, The livers are prepared 24 hours after the last treatment The S9 fractions are produced by dilution of the liver homogenate with a KCI solution (1+3) followed by centrifugation at 9000 g. Aliquots of the supenatant are frozen and stored in ampoules at -80 °C. Small numbers of Ihe ampoules can be kept at -20 °C for up to one week. Each balch of S9 mix is routinely tested wilh 2-aminoanthracene as well as benzo(a}pyrene.

Test concentrations with justification for top dose:

3; 10; 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 non-toxicity to the bacteria.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA 1535, TA 100; without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine

Remarks:

TA 1537,TA98; without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

WP2 uvrA; without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

TA 1535, TA 1537. TA 98, TA 100, WP2 uvrA ; With metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

Evaluation criteria:

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 WP2 uvrA) 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 al 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:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

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

Remarks on result:

other: strain/cell type:

Remarks:

Migrated from field 'Test system'.

No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolite activation. A moderate but dose dependent increase in revertant colony numbers was observed following treatment with FAT 360341G in experiment I in strain TA 1537 in the absence of metabolite activation (S9 mix) and in strain TA 98 in the presence of metabolite activation. In strain TA 1537 the threshold of three times the number of the corresponding solvent control was exceeded at 5000 µg/plate. In strain TA 98 the required threshold of twice the number of the corresponding control was reached at 2500 µg/plate and exceeded at 5000 µg/plate. No comparable increase of the mutation frequency occurred in the other strains. A confirmatory experiment was performed under identically conditions 10 reproduce this minor increase. The results of the confirmatory confirmed the minor increase in the number of the revertants in strain TA 1537 without 89 mix and in strain TA 98 with S9 mix. In this experiment in strain TA 1537 the threshold of three limes the number of the corresponding solvent control was exceeded at 5000 µg/plate. In strain TA 98 the required threshold of twice the number of the corresponding control was reached at 5000 µg/plate.

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

Conclusions:

FAT 36034/G is considered to be mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of FAT 36034/G to induce gene mutations in the plate incorporation test (experiment I) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA as per OECD 471 guideline. 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/Experiment l and l a: 3, 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate.

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments. No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. A moderate but dose dependent increase in revertant colony numbers was observed following treatment with FAT 360341G in experiment in strain TA 1537 in the absence of metabolic activation (S9 mix) and in strain TA 98 in the presence of metabolic activation. In strain TA 1537 the threshold of three times the number of the corresponding solvent control was exceeded at 5000 µg/plate. In strain TA 98 the required threshold of twice the number of the corresponding control was reached at 2500 ug/plate and exceeded at 5000 µg/plate, No comparable increase of the mutation frequency occurred in the other strains. A confirmatory experiment was performed under identical conditions to reproduce this minor increase. The results of the confirmatory confirmed the minor increase in the number of the revertants In strain TA 1537 without S9 mix and in strain TA 98 with S9 mix. In this experiment in strain TA 1537 the threshold of three times the number of the corresponding solvent control was exceeded at 5000 µg/plate. In strain TA 98 the required threshold of mice the number of the corresponding control was reached at 5000 µg/plate. 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 frame shifts in the genome of the strains TA 1537 (without S9 mix) and TA 98 (with S9 mix). Therefore, FAT 36034/G is considered to be mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

Refer chapter 13 for detailed read across justification.

Reason / purpose for cross-reference:

read-across source

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

Additional strain / cell type characteristics:

not specified

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 (without metabolic activation):
0.001, 0.0025, 0.005, 0.010, 0.025, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10 and 0.25 mM
Pre-experiment for experiment II (only without metabolic activation, 20 h long-term exposure assay):
0.001, 0.0025, 0.005, 0.010, 0.025, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10 and 0.25 mM
Experiment I
without and with metabolic activation: 0.005, 0.010, 0.025, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10 and 0.25 mM
Experiment II
without metabolic activation: 0.0025, 0.005, 0.01, 0.02, 0.04, 0.06, 0.08, 0.10, 0.15 and 0.20 mM
and with metabolic activation: 0.025, 0.035, 0.045, 0.055, 0.065, 0.075, 0.085, 0.095, 0.12 and 0.25 mM

Vehicle / solvent:

Vehicle (Solvent) used: DMSO

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: ≥ 0.05 mM; Experiment II without S9: ≥ 0.04 mM; Experiment II with S9: 0.25 mM

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

Based on the read across data generated from the in vitro cell gene mutagenicity test under the experimental conditions, the test item FAT 36034/W TE is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.

Executive summary:

Data on mammalian mutagenicity and clastogenicity was not available for the target substance. To fill the data gaps, read across approach is adapted using similar substance FAT 36034/W. Read-across is claimed basis of structural relationship of the target and the source chemicals. The source chemical, FAT 36034/W TE was assessed for its potential to induce mutations at the HPRT locus using V79 cells of the Chinese Hamster. The selection of the concentrations was based on data from the pre-experiments. Experiment I with and without metabolic activation and experiment II with metabolic activation were performed as a 4 h short-term exposure assay. Experiment II without metabolic activation was performed as 20 h long time exposure assay. The test item was investigated at the following concentrations:

Experiment I

without and with metabolic activation:

0.005, 0.010, 0.025, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10 and 0.25 mM

Experiment II

without metabolic activation:

0.0025, 0.005, 0.01, 0.02, 0.04, 0.06, 0.08, 0.10, 0.15 and 0.20 mM

and with metabolic activation:

0.025, 0.035, 0.045, 0.055, 0.065, 0.075, 0.085, 0.095, 0.12 and 0.25 mM

Precipitation of the test item was noted in experiment I at concentrations of 0.10 mM and higher at the beginning of the treatment and at concentrations of 0.09 mM and higher at the end of the treatment period (without and with metabolic activation). In experiment II no precipitation of the test item was detected without metabolic activation at the beginning of the treatment but at 0.15 mM and higher at the end of the treatment period precipitation was observed. With metabolic activation precipitation of the test item was noted at concentrations of 0.12 mM and higher at the beginning of the treatment and at concentrations of 0.085 mM and higher at the end of the treatment period. Biologically relevant growth inhibition was observed after the treatment with the test item in experiment I without and in experiment II without and with metabolic activation. In experiment I without metabolic activation the relative growth was 36.0 % for the highest concentration (0.25 mM) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 0.25 mM with a relative growth of 77.5 %. In experiment II without metabolic activation the relative growth was 12.2 % for the highest concentration (0.20 mM) evaluated. The highest concentration evaluated with metabolic activation was 0.25 mM with a relative growth of 59.8 %. 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. In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item FAT 36034/W 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

Disperse Red 86:1 was found to be not clastogenic when tested in a micronucleus assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genetic toxicity

The target chemical, Disperse Red 086:1 has been tested positive for mutagenic effects in the Ames assay, while not being clastogenic in an in vivo micronucleus assay. Hence, to complete the data requirement and assessment of its genotoxicity potential, additional study investigating mutagenicity in mammalian cells is required. The source chemical, Disperse Red 086 (FAT 36034/E, purity: 93 %) was found to have weak mutagenic effect in an Ames assay, while it was found to be not mutagenic in the in vitro mammalian cell gene mutation assay. Hence, the data available with the source chemical Disperse Red 86 was used for completion of data requirement as well as to conclude on the genotoxicity potential of Disperse Red 86:1.

 

Bacterial reverse mutation assay:

Disperse Red 86:1 (FAT 36034/G) was assessed to induce gene mutations in the plate incorporation test (experiment I) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and the Escherichia coli strain WP2 uvrA as per OECD Guideline 471. The assay was performed with and without liver microsomal activation. A moderate but dose dependent increase in revertant colony numbers was observed following treatment with FAT 36034/G in experiment in strain TA 1537 in the absence of metabolic activation (S9 mix) and in strain TA 98 in the presence of metabolic activation. A confirmatory experiment was performed under identical conditions to reproduce this minor increase. The results of the confirmatory experiment confirmed the minor increase in the number of the revertants in strain TA 1537 without S9 mix and in strain TA 98 with S9 mix. Therefore, FAT 36034/G is considered to be mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Disperse Red 86:1 (FAT 36034/D) was tested for mutagenic effects in Salmonella typhimurium strains TA 100, TA 1535, TA 102, TA 98 and TA 1537 with and without metabolic activation at the test concentrations of 61.7, 185.2, 555.6, 1666.7 and 5000 µg/plate. When the test substance was exposed to TA 98 with metabolic activation, a slight increase in the number of revertant colonies was seen at 185.2 to 5000 µg/plate in the original and at 555.6 µg/plate (only) in the confirmatory experiment. With strain TA 1537 a similar effect was seen in both original and confirmatory experiments at the concentrations of 555.6 to 5000 µg/plate when tested with metabolic activation and at 1666.7 to 5000 µg/plate when tested without metabolic activation. A marginal increase in the number of revertant colonies occurred on strain TA 102 at the concentrations of 555.6 and 1666.7 µg/plate when tested with metabolic activation in the original experiment only. No effects were observed with the other strains. Based on the results of these experiments and on standard evaluation criteria, it is concluded that FAT 36034/D exerted a slight mutagenic action on strain TA 1537. The metabolites of the test material were weakly mutagenic with strains S. typhimurium TA 98 and TA 1537.

Disperse Red 86 (FAT 36034/E) was tested for mutagenic effects in Salmonella typhimurium strains TA 100, TA 1535, TA 102, TA 98 and TA 1537 with and without metabolic activation at the test concentrations of 61.7, 185.2, 555.6, 1666.7 and 5000 µg/plate. When the test substance was exposed to TA 98 with metabolic activation, a slight increase in the number of revertant colonies was seen at the concentrations of 185.2 to 5000 µg/plate in the original and at 555.6 to 5000 µg/plate in the confirmatory experiment. With strain TA 1537 a similar effect was seen in both original and confirmatory experiments at the concentrations of 555.6 to 5000 µg/plate when tested with metabolic activation and at 1666.7 to 5000 µg/plate (original) when tested without metabolic activation. A marginal increase in the number of revertant colonies occurred with strain TA 1537 at the concentrations of 5000 µg/plate only when tested without metabolic activation in the confirmatory experiment. No effects were observed with the other strains.Based on the results of these experiments and on standard evaluation criteria, it is concluded that FAT 36034/E exerted a marginal mutagenic action on strain TA 1537. The metabolites of the test material were weakly mutagenic with strain S. typhimurium TA 98 and marginally mutagenic with strain TA 1537.

An in vitro study (1979) was performed to assess the potential of Disperse Red 86 (no data on purity) to induce gene mutations in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 when exposed to test concentration of 0, 2, 20, 100 and 300 µg/plate. Under the test conditions, no significant increase in the number of revertant colonies was seen at the test concentrations. Hence, the test substance was considered to be not mutagenic.

In vitro mammalian cell gene mutation assay with the source chemical:

 

Disperse Red 086 was assessed in an in vitro mammalian cell gene mutation assay (HPRT locus), wherein V79 cells were exposed to a concentration range 0.005 to 0.25 mM.In experiment I 0.25 mM (with and without metabolic activation) were selected as the highest concentration. In experiment II 0.2 mM (without metabolic activation) and 0.25 mM (with metabolic activation) were selected as the highest concentrations. Experiment I with and without metabolic activation and experiment II with metabolic activation were performed as a 4 h short-term exposure assay. Experiment II was performed as 20 h long time exposure assay (without metabolic activation). In both experiments with and without metabolic activation, all mutant values of the negative controls, the solvent controls and test item concentrations found were within the historical control data of the test facility. In conclusion, in the described in vitro cell gene mutagenicity test under the experimental conditions reported, the test item FAT 36034/W TE is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.

 

Micronucleus assay:

 

A GLP compliant OECD guideline 474 study was performed to investigate the potential of FAT 36034/D to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The following dose levels of the test article were investigated:

24 h preparation interval: 200, 670 and 2000 mg/kg bw

48 h preparation interval: 2000 mg/kg bw

In comparison to the corresponding vehicle controls there was no substantial enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test article and with any dose level used. Hence, it can be concluded that the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, FAT 36034/D is considered to be non-clastogenic in this micronucleus assay.

Conclusion on genotoxicity

Both Disperse Red 86:1 and Disperse Red 86, were found to have mutagenic action in the bacterial reverse mutation assays, however the source chemical, Disperse Red 86 could not lead to mutagenic action in an in vitro mammalian cell gene mutation assay. Further, Disperse Red 86:1 was found to be not clastogenic when tested in a micronucleus assay. Based on the negative outcome for mutagenicity with the source chemical and clastogenicity with the target chemical, Disperse Red 86:1 is considered to be not genotoxic.

Justification for classification or non-classification

Disperse Red 86:1 was considered to be not genotoxic based on the available information, hence it does not warrant classification for mutagenicity as per the criteria of Regulation (EC) No. 1272/2008.