4-[(1-amino-9,10-dihydro-4-hydroxy-9,10-dioxo-2-anthryl)oxy]-N-(3-ethoxypropyl)benzenesulphonamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Disperse Red 092 was found to be non-mutagenic in a bacterial reverse mutation assay. Hence, in order to fulfil the data requirements of Annex VIII of Regulation (EC) 1907/2006, data from read-across substances has been used. The read-across substance FAT 36034/W was non-mutagenic in a mammalian cell gene mutation assay (HPRT). Hence, on the basis of read-across principles, Disperse Red 092 was considered to be non-mutagenic.

Link to relevant study records

Reference

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:

29 March 2016 to 09 June 2016

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:

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:
PCR92X140707 (China)
- Expiration date of the lot/batch:
14 October 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
2-8 °C, protected from light

Target gene:

The Salmonella strains contain mutations in the histidine operon, thereby imposing a requirement for histidine in the growth medium. These strains contain the deep rough (rfa) mutation, which deletes the polysaccharide side chain from the lipopolysaccharides of the bacterial cell surface. This increases cell permeability of larger substances. The other mutation is a deletion of the uvrB gene, which codes for a protein of the DNA nucleotide excision repair system, resulting in an increased sensitivity in detecting many mutagens. This deletion also includes the nitrate reductase (chi) and biotin (bio) genes (bacteria require biotin for growth). Tester strains TA98 and TA100 contain the R-factor plasmid, pKM101. These strains are reverted by a number of mutagens that are detected weakly or not at all with the non-R-factor parent strains. pKM101 increases chemical and spontaneous mutagenesis by enhancing an error-prone DNA repair system, which is normally present in these organisms. The tester strain Escherichia coli WP2 uvrA carries the defect in one of the genes for tryptophan biosynthesis. Tryptophan-independent mutants (revertants) can arise either by a base change at the site of the original alteration or by a base change elsewhere in the chromosome so that the original defect is suppressed. This second possibility can occur in several different ways so that the system seems capable of detecting all types of mutagens, which substitute one base for another. Additionally, the strain is deficient in the DNA nucleotide excision repair system.

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:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

In the preliminary toxicity assay, the dose levels tested were 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333 and 5000 µg per plate. In the mutagenicity assay, the dose levels tested were 50.0, 150, 500, 1500 and 5000 µg per plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO was the vehicle of choice based on the solubility of the test substance and compatibility with the target cells. The test substance in DMSO formed a clear solution at a concentration of approximately 25 mg/mL and workable suspensions at concentrations of approximately 30 to 250 mg/mL in the solubility test conducted at BioReliance.

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

TA98, TA1535, TA100, TA1537 and WP2 uvrA - WIth S9 activation

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA98 -Without S9 activation

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA100, TA1535 without S9 activation

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA1537 - without S9 activation

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

WP2 uvrA - without S9 activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 to 72 hours

NUMBER OF REPLICATIONS: 1 in the preliminary toxicity assay; 3 in the mutagenicity assay

NUMBER OF CELLS EVALUATED: >/= 0.3 x 10 E8 cells/plate

DETERMINATION OF CYTOTOXICITY
- Method: other: Counting of revertant colony numbers and evaluation of the condition of the bacterial background lawn.

Evaluation criteria:

The revertant colony numbers were determined for each plate (counted either manually or by automatic colony counter). The mean and standard deviation of the number of revertants per plate were calculated and reported. For the test article to be evaluated positive (mutagenic), it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test article as specified below:

Strains TA1535 and TA1537:
Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean vehicle control value.

Strains TA98, TA100 and WP2 uvrA:
Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0-times the mean vehicle control value.

An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response was evaluated as negative if it was neither positive nor equivocal.

Statistics:

According to the test guidelines, the biological relevance of the results is the criterion for the interpretation of the results, and a statistical evaluation of the results is not regarded as necessary.

Key result

Species / strain:

other: TA98, TA1535, TA100, TA1537 and WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Conclusions:

FAT40444/B considered as non-mutagenic with any of the tester strains in either the presence or absence metabolic activation.

Executive summary:

The test substance, FAT40444/B, was tested to evaluate its mutagenic potential by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system. The study was carried out according to OECD 471 guideline. Dimethyl sulfoxide (DMSO) was used as the vehicle. In the preliminary toxicity assay, the dose levels tested were 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333 and 5000 µg per plate. No background lawn toxicity was observed. Precipitate was observed beginning at 667 µg per plate. Based upon these results, the maximum dose tested in the mutagenicity assay was 5000 µg per plate. In the mutagenicity assay, the dose levels tested were 50, 150, 500, 1500 and 5000 µg per plate. No toxicity was observed. Precipitate was observed beginning at 1500 µg per plate. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. The vehicle and positive control values indicate that all criteria for a valid assay were met. Based on the test results, FAT 40444/B was negative for the ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No in vivo genetic toxicity study is available with Disperse Red 092. However, read-across substance FAT 36034/D was found to be non-clastogenic in a micronucleus assay. Hence, on the basis of read across principles, Disperse Red 092 was considered to be not-clastogenic. Currently no in vitro study is available to evaluate effects on chromosomal level in eucaryontic cells. Since in vivo data for one read-across substance are available and in vitro data for a second read-across substance to cover clastogenic effects potentially caused by the test substance the respective in vitro assay is waived for the target substance.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

Refer chapter 13 for detailed read-across justification.

Reason / purpose for cross-reference:

read-across source

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

Based on the read across data generated from the in vivo micronucleus assay with FAT 36034/D, the Disperse Red 092 is also considered as non-clastogenic.

Executive summary:

Data on clastogenicity was not available for the target substance (Disperse Red 092). To fill the data gaps, read-across approach is adapted using similar substance Disperse Red 086 (FAT 36034/D). Read-across is claimed basis of structural relationship of the target and the source chemicals. Read-across substance is FAT 36034/D and have been investigated for clastogenicity. 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 b.w., 48 h preparation interval: 2000 mg/kg b.w.. The highest guideline-recommended dose (2000 mg/kg) was estimated by a pre-experiment to be suitable. The animals expressed slight toxic reactions. After treatment with the test article the number of NCEs was not increased as compared to the corresponding negative controls thus, indicating that FAT 36034/D had no cytotoxic effectiveness in the bone marrow. 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. 20 mg/kg b.w. cyclophosphamide administered was used as positive control which showed a statistically significant increase of induced micronucleus frequency. In conclusion, it can be stated that during the study described and under the experimental conditions reported, the read-across substance did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, the target substance, Disperse Red 092 is also considered to be non-clastogenic in the in-vivo micronucleus assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Bacterial reverse mutation assay with FAT 40444/B

The test substance, FAT40444/B, was tested to evaluate its mutagenic potential by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system according to OECD guideline 471. Dimethyl sulfoxide (DMSO) was used as the vehicle. In the preliminary toxicity assay, the dose levels tested were 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333 and 5000 µg per plate. No background lawn toxicity was observed. Precipitate was observed beginning at 667 µg per plate. Based upon these results, the maximum dose tested in the mutagenicity assay was 5000 µg per plate. In the mutagenicity assay, the dose levels tested were 50.0, 150, 500, 1500 and 5000 µg per plate. No toxicity was observed. Precipitate was observed beginning at 1500 µg per plate. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. The vehicle and positive control values indicate that all criteria for a valid assay were met. These results indicate FAT40444/B was negative for the ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system.

 

Mammalian cell gene mutation assay in vitro with FAT 36034/W (read across)

In a mammalian cell gene mutation assay (HPRT locus),V79 cells cultured in vitro were exposed to a read-across substance, FAT 36034/W at, concentrations up to 0.005, 0.25 mM (without and with metabolic activation). In experiment I without metabolic activation the highest mutation rate (compared to the solvent control values) of 1.54 was found at a concentration of 0.09 mM with a relative growth of 59.0%. In experiment I with metabolic activation the highest mutation rate (compared to the solvent control values) of 1.03 was found at a concentration of 0.06 mM with a relative growth of 92.8 %. In experiment II without metabolic activation the highest mutation rate (compared to the solvent control values) of 1.30 was found at a concentration of 0.01 mM with a relative growth of 75.7 %. In experiment II with metabolic activation the highest mutation rate (compared to the solvent control values) of 2.28 was found at a concentration of 0.085 mM with a relative growth of 78.2 %. The positive controls did induce the appropriate response. There was no evidence of a concentration related positive response of induced mutant colonies over background. Hence, the read across substance, FAT 36034/W, is considered to be non-mutagenic in the HPRT assay using V79 cells of the Chinese Hamster.

 

Micronucleus assay in vivo with FAT 36034/D (read-across)

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 b.w., 48 h preparation interval: 2000 mg/kg b.w. The highest guideline-recommended dose (2000 mg/kg) was estimated by a pre-experiment to be suitable. The animals expressed slight toxic reactions. After treatment with the test article the number of NCEs was not increased as compared to the corresponding negative controls thus indicating that FAT 36034/D had no cytotoxic effectiveness in the bone marrow. 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. 20 mg/kg b.w. cyclophosphamide administered was used as positive control which showed a statistically significant increase of induced micronucleus frequency. In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, FAT 36034/D is considered to be non-clastogenic in this micronucleus assay.

 

FAT 40444/B was found to be non-mutagenic in a bacterial reverse mutation assay. Read-across substance, FAT 36034/W was found to be non-mutagenic in a mammalian cell gene mutation assay in vitro (HPRT), while read across substance, FAT 41034/A was non-clastogenic in a chromosomal aberration assay in vitro. Another read across substance, FAT 36034/A was non-clastogenic in a micronucleus assay in vivo. Hence, based on the negative bacterial reverse mutation assay with the substance and negative results from different read across substances in a mammalian cell gene mutation assay, a chromosomal aberration assay in vitro and an in-vivo micronucleus assay, the substance FAT 40444/B was considered to be not-genotoxic.

Justification for classification or non-classification

Based on the available genotoxicity study information, Disperse Red 092 does not considered to be classified for genotoxicity according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.