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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in vitro:
The given test material Chrysoidin shows positive gene toxicity in vitro result in the presence of S9 metabolic activation system and negative result in the absence of S9 metabolic activation system.

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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
Data is from peer reviewed journal
Qualifier:
according to
Guideline:
other: as mentioned below
Principles of method if other than guideline:
Gene toxicity in vitro study was performed on the Salmonella typhimurium TA 1538 strain to evaluate the mutagenic effect of the test material Chrysoidin
GLP compliance:
not specified
Type of assay:
bacterial gene mutation assay
Target gene:
AMES assay
Salmonella typhimurium TA 1538 strain
Species / strain / cell type:
S. typhimurium TA 1538
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Test concentrations with justification for top dose:
50, 100 µg/plate
Vehicle / solvent:
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: No data available
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: Acetylaminofluorene (5 and 10 µg/plate)
Details on test system and experimental conditions:
Details on test system and conditions
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period:
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data available
- Fixation time (start of exposure up to fixation or harvest of cells): No data available

SELECTION AGENT (mutation assays): No data available
SPINDLE INHIBITOR (cytogenetic assays): No data available
STAIN (for cytogenetic assays): No data available

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: No data available

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data available

OTHER EXAMINATIONS:
- Determination of polyploidy: No data available
- Determination of endoreplication: No data available
- Other:

OTHER: No data available
Evaluation criteria:
Numbers of revertants on test plates greater than 30 are classified as being significantly mutagenic
Statistics:
No data available
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
Genotoxicity: Positive
Concentration His+ revertants/plate
Crude Purified
50 931 867
100 1260 1312

Negative
Concentration His+ revertants/plate
Crude Purified
50 11 -
100 11 -
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: Bacterial strain used
Conclusions:
The given test material Chrysoidin shows positive gene toxicity in vitro result in the presence of S9 metabolic activation system and negative result in the absence of S9 metabolic activation system.
Executive summary:

Gene toxicity in vitro study was performed on theSalmonella typhimurium TA 1538 strain to evaluate the mutagenic effect of the test material Chrysoidin.

Chrysoidin was used at a concentration of 50 and 100 µg/ plate. The test material was purified and retested for mutagenicity again.

Crude chrysoidin induced 931 his+ revertants at 50 µg and 1260 his+ at 100 µg per plate while the purified material induced 867 and 1312 his+ revertants respectively. It was noticed on plates containing dye and the liver enzyme preparation that the colour remaining after 48 h incubation was less than on plates without the liver enzyme. Since reduction of the azo group leads to loss of dye colouration it was presumed that the liver enzyme catalysed this reaction probably through liver azo-reductase, an NADPH2 requiring enzyme. Chrysoidin shows positive gene toxicity in vitro result in the presence of S9 metabolic activation system and negative result in the absence of S9 metabolic activation system.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Genetic toxicity in vitro:
Different experimental studies from peer reviewed journals were reviewed for Genetic toxicity in vitro endpoint for the test substance 4-(phenyldiazenyl)benzene-1,3-diamine and are presented below as key and supporting studies:

Gene toxicity in vitro study was performed on the Salmonella typhimurium TA 1538 strain (Mutation Research, 44 (1977) 9-19) to evaluate the mutagenic effect of the test material Chrysoidin. Chrysoidin was used at a concentration of 50 and 100 µg/ plate. The test material was purified and retested for mutagenicity again. Crude chrysoidin induced 931 his+ revertants at 50 µg and 1260 his+ at 100 µg per plate while the purified material induced 867 and 1312 his+ revertants respectively. It was noticed on plates containing dye and the liver enzyme preparation that the colour remaining after 48 h incubation was less than on plates without the liver enzyme. Since reduction of the azo group leads to loss of dye colouration it was presumed that the liver enzyme catalysed this reaction probably through liver azo-reductase, an NADPH2 requiring enzyme. Chrysoidin shows positive gene toxicity in vitro result in the presence of S9 metabolic activation system and negative result in the absence of S9 metabolic activation system.

Supporting S. typhimurium/mammalian microsomal pre-incubation mutagenicity assay was performed on the S. typhimuriumtester strains TA100 and TA98 to detect the mutagenic potential of the test material Chrysoidine Y (Carcinogenesis vol.7 no. 11 pp.1921-1923, 1986). The test was performed in the presence of three types of metabolic activation system including- phenobarbitone – induced rat liver S9 metabolic activation system, phenobarbitone – uninduced rat liver S9 metabolic activation system, With uninduced human liver S9 metabolic activation system. Positive results were noted for all the test systems in the presence of the metabolic activation system specified. The ratio of mutagenic potency is greater in the phenobarbital-induced system than in either of the uninduced systems suggesting that a phenobarbital-inducible form of cytochrome P450 is involved in the activation of the dye. Chrysoidine Y is positive to induce mutation in theS. typhimurium/mammalian microsomal pre-incubation mutagenicity assay performed on the S. typhimurium tester strains TA100 and TA98 specifically in the phenobarbitone – induced rat liver S9 metabolic activation system.

Further, Salmonella/mammalian-microsome mutagenicity test was performed for the test compound 2, 4-diamino-azobenzene in different labs A-H with and without S9 metabolic activation system (Mutation Research, 97 (1982) 429-435). The doses were chosen according to toxicity or solubility of the compound but were not higher than 2500 µg/plate. 5 consecutive doses were used each generated by a 5-fold dilution of higher dose. Five different solvents were used by different labs including water, acetone, methanol, ethanol, and DMSO. Positive control 9, 10-dimethylanthracene and negative control anthracene were also incorporated in the test. As per the results obtained,the test material2, 4-diamino-azobenzene is Positive (With metabolic activation system S9) and Negative (Without metabolic activation system S9) in the Salmonella/mammalian-microsome mutagenicity test performed in the labs A-H.

Further supporting, AMES assay was performed to evaluate the mutagenic nature of the test compound 2, 4 Diaminoazobenzene (Mutat Res 85:260, 1981). The test compound 2,4 Diaminoazobenzene has the ability to cause reversion in theSalmonella typhimuriumstrains TA98, TA 100 and TA 1537 and hence is found to be mutagenic in vitro.

In support of all the above data, the Salmonella/S9 mutagenicity assay was performed (Toxicology Letters, 58 (1991) 43-50) to evaluate the mutagenic nature of the test compound chrysoidine R with or without the addition of β-glucuronidase (βG). Chrysoidine R was dissolved in dimethyl sulphoxide (DMSO) and added (20 µL) at a concentration of 5-80 µg/plate. The test compound was found to increase the number of revertant colonies per plate and hence is mutagenic in vitro.

Thus, considering the key and various supporting studies for the substance 4-(phenylazo)benzene-1,3-diamine (CAS No. 495-54-5), it was concluded that the substance is genetically toxic and hence it can be classified under Mutagenic category 2 as per the criteria of CLP regulation.

Justification for classification or non-classification

Considering the key and various supporting studies for the substance 4-(phenylazo)benzene-1,3-diamine (CAS No. 495-54-5), it was concluded that the substance is genetically toxic and hence it can be classified under Mutagenic category 2 as per the criteria of CLP regulation.