Phenol, 4-[[4-(dimethylamino)phenyl]azo]-, reaction products with sodium sulfide (Na2(Sx)), leuco derivatives

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the results of an OECD 471 study, the test substance is considered to be non-mutagenic with and without metabolic activation in bacteria.

Based on the results of an OECD 476 study with the read across substance (CAS: 1040873 -93 -5), the test substance is considered to be non-mutagenic in Chinese hamster ovary cells.

Based on the results of an OECD 473 study with the read across substance (CAS: 1040873 -93 -5), the test substance is considered to be non-clastogenic in Chinese Hamster lung V79 cells.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 August 2010 - 04 October 2010

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:

1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

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:

Phenobarbital/ß-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Pre-Experiment/Experiment I and II: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate (top concentration compliant to guideline)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Sufficient test item solubility and non-toxicity to tester strains

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- Pre-experiment/Experiment I: in agar (plate incorporation)
- Experiment II: preincubation

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: triplicates

DETERMINATION OF CYTOTOXICITY
- Reduction in the number of revertants (below the indication factor of 0.5)
- Reduction of background lawn

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 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:

Not performed (not mandatory for this test system).

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test item precipitated in the overlay agar on the incubated agar plates from 2500 μg/plate up to 5000 μg/plate in both experiments with and without S9 mix. The undissolved particles had no influence on the data recording.

see "attached background material"

Conclusions:

The test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Executive summary:

This study according to OECD guideline 471 was performed to investigate the potential of the test item (commercial formulation of Leuco Sulphur Blue 9, containing 15% dyestuff) to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both 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 I and II: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate. The plates incubated with the test item showed reduced background growth in all strains. Precipitation and/or toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains at concentrations of 1000 µg/plate and/or above. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. The test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

Please refer to the attached read-across justification in section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

Sub-line (K1)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Evidence of toxicity was seen at the highest tested concentration with the test item in presence and absence of metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

Please refer to the attached read-across justification in section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

OECD 471:

The study according to OECD guideline 471 was performed to investigate the potential of the test item (commercial formulation of Leuco Sulphur Blue 11, containing 15% dyestuff) to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both 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 I and II: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate. The plates incubated with the test item showed reduced background growth in all strains. Precipitation and/or toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains at concentrations of 1000 µg/plate and/or above. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. The test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Even though the commercial formulation was tested, containing 15% dyestuff compared to 88 -95% dyestuff present in the REACh registration sample, the study is suitable for the evaluation of the REACh registration substance since the test item precipitated and induced cytotoxicity at high concentrations.

OECD 476:

The read across substance (CAS: 1040873 -93 -5) suspended in DMSO, was tested in a Mammalian Gene Mutation Test in CHO-K1 cells according to OECD guideline 476. The following concentrations were selected on the basis of a pre-test on cytotoxicity without and with metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver: 39.1, 78.2, 156.3, 234.4 and 312.5 µg/mL (without S9-mix) and 39.1, 78.2, 156.3, 312.5, 625 and 937.5 µg/mL (with S9-mix).Phenotypic expression was evaluated up to 8 days following exposure.There was no precipitation of the test item at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested. In the absence and presence of metabolic activation clear cytotoxicity (survival between 17-19 %) of the test item was observed at the highest concentration applied (312.5 µg/mL in the absence and 937.5 µg/mL in the presence of S9 mix). In both experimental parts, there were no increases in mutation frequency when compared to the concurrent solvent control and the laboratory historical control data at any concentration tested in the absence and presence of metabolic activation. All results were inside the distribution of the historical negative control data (based 95% control limit).The mutation frequency found in the solvent controls was in the 95% confidence interval of the historical control data. The concurrent positive controls ethyl methanesulfonate (1.0 µL/mL) and7, 12-dimethyl benzanthracene (20 µg/mL) caused the expected biologically relevant increases of cells with mutation frequency as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.The test itemtested up to cytotoxicconcentrationswith and without metabolic activationover a 5 hour treatment period did not induce statistically significant and biologically relevant increases in mutant frequency. It is concluded that the test item was not mutagenic in this in vitro mammalian cell gene mutation test performed with Chinese hamster ovary cells.

OECD 473:

The read across substance (CAS: 1040873 -93 -5) suspended in DMSO was tested in a chromosome aberration assay in V79 cells in two independent experiments according to OECD guideline 473. For the cytogenetic experiments the following concentrations were selected on the basis of a pre-test on (without and with metabolic activation using rodent S9 mix): 31.3, 62.5, 125 and 250g/mL test item for Experiment A with 3/20 h treatment/sampling time and Experiment B with 3/28 h treatment/sampling time and 7.8, 15.7, 31.3and 62.5g/mL test item for Experiment B with 20/20 h and 20/28 h treatment/sampling time. Following treatment and recovery the cells were exposed to the spindle inhibitor colchicine (0.2 µg/mL) 2.5 hours prior to harvesting. Harvested cells were treated with fixative for ca. 10 minutes before being placed on slides and stained. In each experimental group duplicate cultures were evaluated for cytogenetic damage (150 metaphases per culture). No precipitation of the test item was observed at any of the applied concentrations. There were no relevant changes in pH or osmolality after treatment with the test item. Clear cytotoxicity of about 50% was observed after test item treatment in all experimental parts. No relevant increases in cells carrying structural chromosomal aberrations were observed, neither in the absence nor in the presence of metabolic activation. In experiment A in the absence of metabolic activation, two values were slightly above the 95% control limits of the historical control data. However, no statistical significant differences were observed after test item treatment when compared to the concurrent solvent as well as the historical control groups. In addition, no dose-response relationship was observed and therefore, the findings were not considered as being biologically relevant. There were no biologically relevant increases in the rate of polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation. The number of aberrations found in the solvent controls was in the range of the historical laboratory control data. The concurrent positive controls ethyl methanesulphonate (0.4 and 1.0 L/mL) and cyclophosphamide (5 g/mL) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid. In conclusion, the test item did not induce structural chromosome aberrations in Chinese Hamster lung V79 cells, when tested up to cytotoxic concentrations in the absence and presence of metabolic activation. Thus, the test item is considered as being non-clastogenic in this system.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genetic toxicity, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the twelfth time in Regulation (EU) No 2019/521.