Hydrogen [4-[4-(diethylamino)-2',4'-disulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene]diethylammonium, aluminium salt

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the prediction done using the OECD QSAR toolbox version 3.4 with log kow as the primary descriptor and considering the five closest read across substances, gene mutation was predicted for Hydrogen [4-[4-(diethylamino)-2',4'-disulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene] diethylammonium, aluminium salt. The study assumed the use of Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 with and without S9 metabolic activation system. Hydrogen [4-[4-(diethylamino)-2',4'-disulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene] diethylammonium, aluminium salt was predicted to not induce gene mutation in Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the presence and absence of S9 metabolic activation system and hence, according to the prediction made, it is not likely to classify as a gene mutant in vitro.

Based on the predicted result it can be concluded that the substance is considered to be not toxic as per the criteria mentioned in CLP regulation.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with limited documentation / justification

Justification for type of information:

Data is from OECD QSAR Toolbox version 3.4 and the spporting QMRF report has been attached

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

Prediction is done using OECD QSAR Toolbox version 3.4, 2017

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name of the test chemical: Hydrogen [4-[4-(diethylamino)-2',4'-disulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene]diethylammonium, aluminium salt
- Molecular formula: C27H32N2O6S2.1/3Al
- Molecular weight: 1658.0277 g/mol
- Smiles Notation: CCN(CC)c1ccc(cc1)C(=C2C=CC(=[N+](CC)CC)C=C2)c3ccc(cc3S(=O)(=O)[O-])S(=O)(=O)[O-].CCN(CC)c1ccc(cc1)C(=C2C=CC(=[N+] (CC)CC)C=C2)c3ccc(cc3S(=O)(=O)[O-])S(=O)(=O)[O-].CCN(CC)c1ccc(cc1)C(=C2C=CC(=[N+](CC)CC)C=C2)c3ccc(cc3S(=O)(=O)[O-])S(=O)(=O)[O-].[Al+3]
- InChI: 1S/3C27H32N2O6S2.Al/c3*1-5-28(6-2)22-13-9-20(10-14-22)27(21-11-15-23(16-12-21)29(7-3)8-4)25-18-17-24(36(30,31)32)19-26(25)37(33,34) 35;/h3*9-19H,5-8H2,1-4H3,(H-,30,31,32,33,34,35);/q;;;+3/p-3
- Substance Type: Organic
- Physical State: Solid

Target gene:

Histidine

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

S9 metabolic activation system

Test concentrations with justification for top dose:

No data

Vehicle / solvent:

No data

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Details on test system and experimental conditions:

No data

Rationale for test conditions:

No data

Evaluation criteria:

Prediction is done considering a dose dependent increase in the number of revertants/plate

Statistics:

No data

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

No data

Remarks on result:

no mutagenic potential (based on QSAR/QSPR prediction)

The prediction was based on dataset comprised from the following descriptors: "Gene mutation"
Estimation method: Takes highest mode value from the 5 nearest neighbours
Domain  logical expression:Result: In Domain

(((((("a" or "b" or "c" or "d" or "e" )  and ("f" and ( not "g") )  )  and ("h" and ( not "i") )  )  and "j" )  and ("k" and ( not "l") )  )  and ("m" and "n" )  )

Domain logical expression index: "a"

Referential boundary: The target chemical should be classified as Alkene AND Ammonium salt AND Aromatic amine AND Aryl AND Sulfonic acid by Organic Functional groups

Domain logical expression index: "b"

Referential boundary: The target chemical should be classified as Alkene AND Ammonium salt AND Aromatic amine AND Aryl AND Overlapping groups AND Sulfonic acid by Organic Functional groups (nested)

Domain logical expression index: "c"

Referential boundary: The target chemical should be classified as Aliphatic Carbon [CH] AND Aliphatic Carbon [-CH2-] AND Aliphatic Carbon [-CH3] AND Aliphatic Nitrogen, one aromatic attach [-N] AND Amino, aliphatic attach [-N<] AND Aromatic Carbon [C] AND Hydroxy, sulfur attach [-OH] AND Miscellaneous sulfide (=S) or oxide (=O) AND Olefinic carbon [=CH- or =C<] AND Suflur {v+4} or {v+6} AND Sulfinic acid [-S(=O)OH] AND Sulfonate, aromatic attach [-SO2-O] by Organic functional groups (US EPA)

Domain logical expression index: "d"

Referential boundary: The target chemical should be classified as Amine AND Anion AND Aromatic compound AND Cation AND Sulfonic acid AND Sulfonic acid derivative AND Tertiary amine AND Tertiary mixed amine by Organic functional groups, Norbert Haider (checkmol)

Domain logical expression index: "e"

Similarity boundary:Target: CCN(CC)c1ccc(C(=C2C=CC(=N{+}(CC)CC)C=C2)c2ccc(S(=O)(=O)O{-})cc2S(O)(=O)=O)cc1
Threshold=50%,
Dice(Atom centered fragments)
Atom type; Count H attached; Hybridization

Domain logical expression index: "f"

Referential boundary: The target chemical should be classified as No alert found by DNA alerts for AMES by OASIS v.1.4

Domain logical expression index: "g"

Referential boundary: The target chemical should be classified as AN2 OR AN2 >>  Michael-type addition, quinoid structures OR AN2 >>  Michael-type addition, quinoid structures >> Quinoneimines OR Radical OR Radical >> ROS formation after GSH depletion (indirect) OR Radical >> ROS formation after GSH depletion (indirect) >> Quinoneimines by DNA alerts for AMES by OASIS v.1.4

Domain logical expression index: "h"

Referential boundary: The target chemical should be classified as No alert found by in vitro mutagenicity (Ames test) alerts by ISS

Domain logical expression index: "i"

Referential boundary: The target chemical should be classified as alpha,beta-unsaturated carbonyls OR Aromatic diazo OR Aromatic mono-and dialkylamine by in vitro mutagenicity (Ames test) alerts by ISS

Domain logical expression index: "j"

Referential boundary: The target chemical should be classified as Not bioavailable by Lipinski Rule Oasis ONLY

Domain logical expression index: "k"

Referential boundary: The target chemical should be classified as No alert found by DNA binding by OASIS v.1.4

Domain logical expression index: "l"

Referential boundary: The target chemical should be classified as Non-covalent interaction OR Non-covalent interaction >> DNA intercalation OR Non-covalent interaction >> DNA intercalation >> Fused-Ring Primary Aromatic Amines OR Radical OR Radical >> Radical mechanism via ROS formation (indirect) OR Radical >> Radical mechanism via ROS formation (indirect) >> Fused-Ring Primary Aromatic Amines OR SN1 OR SN1 >> Nucleophilic attack after metabolic nitrenium ion formation OR SN1 >> Nucleophilic attack after metabolic nitrenium ion formation >> Fused-Ring Primary Aromatic Amines by DNA binding by OASIS v.1.4

Domain logical expression index: "m"

Parametric boundary:The target chemical should have a value of log Kow which is >= -2.41

Domain logical expression index: "n"

Parametric boundary:The target chemical should have a value of log Kow which is <= 2.11

Conclusions:

Hydrogen [4-[4-(diethylamino)-2',4'-disulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene]diethylammonium, aluminium salt was predicted to not induce gene mutation in Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the presence and absence of S9 metabolic activation system and hence, according to the prediction made, it is not likely to classify as a gene mutant in vitro.

Executive summary:

Based on the prediction done using the OECD QSAR toolbox version 3.4 with log kow as the primary descriptor and considering the five closest read across substances, gene mutation was predicted for Hydrogen [4-[4-(diethylamino)-2',4'-disulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene]diethylammonium, aluminium salt. The study assumed the use of Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 with and without S9 metabolic activation system. Hydrogen [4-[4-(diethylamino)-2',4'-disulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene]diethylammonium, aluminium salt was predicted to not induce gene mutation in Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the presence and absence of S9 metabolic activation system and hence, according to the prediction made, it is not likely to classify as a gene mutant in vitro.

Based on the predicted result it can be concluded that the substance is considered to be not toxic as per the criteria mentioned in CLP regulation.

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:

Prediction model based estimation and data from read across chemicals have been reviewed to determine the mutagenic nature of

Hydrogen [4-[4-(diethylamino)-2',4'-disulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene] diethylammonium, aluminium salt. The studies are as mentioned below:

Based on the prediction done using the OECD QSAR toolbox version 3.4 with log kow as the primary descriptor and considering the five closest read across substances, gene mutation was predicted for Hydrogen [4-[4-(diethylamino)-2',4'-disulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene] diethylammonium, aluminium salt. The study assumed the use of Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 with and without S9 metabolic activation system. Hydrogen [4-[4-(diethylamino)-2',4'-disulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene]diethylammonium, aluminium salt was predicted to not induce gene mutation in Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the presence and absence of S9 metabolic activation system and hence, according to the prediction made, it is not likely to classify as a gene mutant in vitro.

Bonin et al (Mutation Research, 1981) performed gene mutation toxicity study for >99% structurally and functionally similar read across chemical.

Salmonella / mammalian microsome mutagenicity assay was performed to study the mutagenic potential of Patent Blue V (RA CAS no 129 -17 -9; IUPAC name: hydrogen [4-[4-(diethylamino)-2',4'-disulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene]diethylammonium, sodium salt

) both in the presence and absence of metabolic activator S9 mix. To each 2 ml of top agar at 42°C was added 100 µL bacterial broth culture, 100 µL test compound dissolved in DMSO various concentrations ranging from 0, 32, 100, 320, 1000 µg/plate, and 500 µL S9 mix as required. Plates were incubated at 37°C for 72 hrs before counting his⁺revertant colonies and each dose point was determined from at least two plates, unless indicated otherwise. Criteria for mutagenicity were (a) a dose-response and, (b) reproducibility of the result. Dose-responses were not always evident at concentratrations selected for initial testing. Paten Blue V did induce mutagenicity in the Salmonella typhimurium TA98, TA100, TA1535, TA1537 and TA1538 strains in the presence and absence of S9 mix and hence it is not likely to classify as a gene mutant.

Muzall and Cook (Mutation Research, 1979) peformed gene mutation toxicity for 80 -90% structurally and functionally similar read across chemical FD and C Red no. 2 (RA CAS no 915 -67 -3; IUPAC name: trisodium (4E)-3-oxo-4-[2-(4-sulfonatonaphthalen-1-yl)hydrazin-1-ylidene]-3,4-dihydronaphthalene-2,7-disulfonate). Spot test was performed at dose levels from 10-250 mg using Salmonella typhimurium strain TA98, TA1537, TA100, TA1535 with and without S9 metabolic activation system. Captan was used as positive control chemical and the solvent control used was DMSO. Mutagenicity was indicated by a clustering of revertant colonies directly around the test material or at the edge of the inhibitory zone. FD&C Red No. 2 did not induce clustering of revertant colonies directly around the test material or at the edge of the inhibitory zone using Salmonella typhimurium strain TA98, TA1537, TA100, TA1535 in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

In the same study by Muzall and Cook, Gene mutation toxicity study was performed to determine the mutagenic nature of structurally and functionally similar read across chemical FD&C Red No. 2 (RA CAS no 915 -67 -3; IUPAC name: trisodium (4E)-3-oxo-4-[2-(4-sulfonatonaphthalen-1-yl)hydrazin-1-ylidene]-3,4-dihydronaphthalene-2,7-disulfonate). The study was performed as per the plate incorporation assay using Salmonella typhimurium strain TA98, TA1537, TA100, TA1535 with and without S9 metabolic activation system. The 2 ml of liquid top agar was cooled to 45°C and 0.1 ml of a broth cultureof microorganism and test substance in volumes of≤0.4 ml of DMSO was added prior to placing on minimal agar plates. The plates were incubated for 48 h at 37°C and the colonies which reverted to the prototroph were counted and compared to counts on the control plate (containing no test substance) to demonstrate mutagenicity or toxicity. Materials which caused a 2-fold increase of revertants, as compared to the number of spontaneous revertants on the control plates, were denoted as mutagens. Those which reduced the number of revertants were considered inhibitory. FD&C Red No. 2 did not result in a 2-fold increase in the number of revertants as compared to the number of spontaneous revertants on the control plates in Salmonella typhimurium strain TA98, TA1537, TA100, TA1535 in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

In another study for structurally and functionally similar read across chemical, Ishidate et al (Food and chemical toxicology, 1984) performed gene mutation toxicity study to determine the mutagenic nature of Food red 102 (RA CAS no 2611 -82 -7; IUPAC name: 1,3-Naphthalenedisulfonic acid, 7-hydroxy-8-((4-sulfo-1-naphthalenyl)azo)-, trisodium salt). The study was performed using S. typhimurium strains TA92, TA1535, TA100, TA1537, TA94 and TA98 with and without S9 metabolic activation system. The test was performed as per the preincubation assay at six different concentrations with 5 mg/plate being the maximum concentration. The chemical was dissolved in phosphate buffer. Preincubation was performed for 20 mins and the exposure duration was for 48 hrs. The result was considered positive if the number of colonies found was twice the number in the control (exposed to the appropriate solvent or untreated). Food red 102 failed to induce a doubling of revertant colonies over the control using S. typhimurium strains TA92, TA1535, TA100, TA1537, TA94 and TA98 in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

In the same study by Ishidate et al, Chromosomal aberration study was performed to determine the mutagenic nature of Food red 102 (RA CAS no 2611 -82 -7; IUPAC name:1,3-Naphthalenedisulfonic acid, 7-hydroxy-8-((4-sulfo-1-naphthalenyl)azo)-, trisodium salt). The cells were exposed to the test material at three different doses with 1 mg/mL being the maximum concentration for 24 and 48 hr. Colcemid (final concn 0.2µg/ml) was added to the culture 2 hr before cell harvesting. The cells were then trypsinized and suspended in a hypotonic KCI solution (0.075 M) for 13 min at room temperature. After centrifugation the cells were fixed with acetic acid-methanol (1:3, v/v) and spread on clean glass slides. After air-drying, the slides were stained with Giemsa solution for 12-15 min. A hundred well-spread metaphases were observed under the microscope. In the present studies, no metabolic activation systems were applied. The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate. Untreated cells and solvent-treated cells served as negative controls, in which the incidence of aberrations was usually less than 3.0%. The results were considered to be negative if the incidence was less than 4.9%, equivocal if it was between 5.0 and 9.9%, and positive if it was more than 10.0%. Food red 102 did not induce chromosomal aberration in chinese hamster fibroblast cell line CHL and hence is not likely to classify as a gene mutant in vitro.

Based on the data availble for the target chemical and its read across, Hydrogen [4-[4-(diethylamino)-2',4'-disulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene] diethylammonium, aluminium salt does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant.

Justification for classification or non-classification

Based on the data availble for the target chemical and its read across, Hydrogen [4-[4-(diethylamino)-2',4'-disulphonatobenzhydrylidene]cyclohexa-2,5-dien-1- ylidene] diethylammonium, aluminium salt (CAS no 15876 -40 -1) does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant.