Phenol, 4-(phenylamino)-, sulfurized, leuco deriv.

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test:

In a bacterial reverse mutation assay in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli strain WP2 uvr no mutagenicity was observed up to the highest required test item concentration in the absence and presence of metabolic activation.

Chromosomal aberration test:

The test substance did not reveal any relevant increases in structural and numerical chromosomal aberrations in peripheral human lymphocytes up to the highest required concentration in the absence and presence of metabolic activation.

HPRT test:

No relevant increases in mutation frequencies were observed after test substance treatment up to the highest required concentration in the absence and presence of metabolic activation in a gene mutation assay at the hprt locus using V79 cells.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

12-Jun-2012 to 14-Sep-2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: Cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

- Type and identity of media:
Blood samples
Blood samples were collected by venapuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.
- Culture medium
Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 µg/mL respectively) and 30 U/mL heparin.
- Lymphocyte cultures
Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 ml (9 mg/mL) phytohaemagglutinin was added.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: not applicable, immediately after blood collection lymphocyte cultures were started.
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: not applicable, immediately after blood collection lymphocyte cultures were started.

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone.

Test concentrations with justification for top dose:

Dose range finding test:
Without and with S9-mix, 3hr exposure; 24 hr fixation: 3, 10, 33, 100 and 333 µg/mL
Without S9-mix, 24/48hr exposure; 24/48 hr fixation: 3, 10, 33, 100, 333 and 1000 µg/mL

First cytogenetic test:
Without and with S9-mix, 3 h exposure time, 24 h fixation time: 17, 50 and 167 µg/mL

Second cytogenetic test:
Without S9-mix, 24 hr exposure; 24 hr fixation: 10, 333 and 1000 µg/mL
Without S9-mix, 48 hr exposure; 48 hr fixation: 10, 100 and 333 µg mL
With S9-mix, 3 hr exposure; 48 hr fixation: 100, 333 and 1000 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:
In the dose range finding study, C.I. Leuco Sulphur Brown 96 was soluble in dimethyl sulfoxide at concentrations of 10 mg/ml and below but formed a suspension at concentrations of 33 mg/ml and above.

At request of the sponsor, the vehicle to be used in the cytogenetic assays was dimethylformamide. C.I. Leuco Sulphur Brown 96 was soluble in dimethylformamide at concentrations of 6.6 mg/ml and below but formed a suspension at concentrations of 20 mg/ml and above.

- Justification for choice of solvent/vehicle: A homogeneous suspension could be obtained in dimethylformamide and dimethylformamide is accepted and approved by authorities and international guidelines

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hr
- Exposure duration: 3 hr (with and without S9-mix), 24 and 48 hr (without S9-mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 and 48 hr

SPINDLE INHIBITOR (cytogenetic assays): colchicine
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicates in two independent experiments

NUMBER OF CELLS EVALUATED: 100 metaphase chromosome spreads per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of each culture was determined by counting the number of metaphases per 1000 cells

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.

A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.

Statistics:

The incidence of aberrant cells (cells with one or more chromosome aberrations, gaps included or excluded) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics.

Key result

Species / strain:

lymphocytes: human pheripheral blood

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No

- Precipitation: Precipitation in the exposure medium was observed at dose levels of 333 µg/ml and above in the dose range finding test and the first cytogenicity test. Precipitation in the exposure medium was observed at dose levels of 1000 µg/ml and above in the second cytogenicity test.


RANGE-FINDING/SCREENING STUDIES:
- No toxicity was observed up to and including the highest precipitating tested dose

COMPARISON WITH HISTORICAL CONTROL DATA:
- The number of cells with chromosome aberrations found in the solvent and positive control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide induced appropriate responses.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- The test was scored up to or beyond dose levels with precipitate. Except in the second experiment in the presence of S9-mix, in this part of the experiment appropriate toxicity was reached.

Conclusions:

The test substance did not reveal any relevant increases in structural and numerical chromosomal aberrations in peripheral human lymphocytes up to the highest required concentration in the absence and presence of metabolic activation.

Executive summary:

In a chromosomal aberration assay using peripheral human lymphocytes C.I. Leuco Sulphur Brown 96 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently repeated experiments up to a precipitating concentration range with a top dose of 1000 µg/mL. No cytotoxicity was observed after treatment with the substance.

The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system functioned properly. 

No effects of C.I. Leuco Sulphur Brown 96 on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix.

Therefore it can be concluded that C.I. Leuco Sulphur Brown 96 does not disturb mitotic processes and cell cycle progression and does not induce structural and numerical chromosome aberrations under the experimental conditions described.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

14 February 2017 until 10 March 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

Identification: Leuco Sulphur Brown 96
CAS No.: 85736-99-8
Batch: 106152313
Molecular Weight: Not applicable
Purity: Approx. 20% *
Expiry / Retest Date: 28 July 2021
Stability in Solvent: Not indicated by the sponsor
Storage Conditions: At room temperature, light protected
Appearance: Solid (presscake)

* Dose calculation adjusted to purity

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/Beta-Naphtoflavone induced Rat liver S9

Test concentrations with justification for top dose:

without metabolic activation: 39.1; 78.1; 156.3; 312.5; 625.0, 1250 µg/mL
with metabolic activation: 19.5; 39.1; 78.1; 156.3; 312.5, 625.0 µg/mL

The concentration range of the main experiment was based on precipitation observed in the pre-experiment and therefore 1250 µg/mL was chosen as top concentration in the absence of metabolic activation and 625.0 µg/mL as top concentration in the presence of metablic activation in the main experiment.
To overcome problems with possible deviations in toxicity and precipitation the main experiment was started with more than four concentrations.
The cultures at the highest concentration with (625.0 µg/mL) and without (1250.0 µg/mL) metabolic activation were not continued to avoid analysis of too many precpitating concentrations.

Vehicle / solvent:

Concurrent solvent controls (deionised water (local tap water, deionised at Envigo CRS GmbH)) were performed.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: Experiment I: 4 hours with and without metabolic activation
- Expression time (cells in growth medium): 72 hours
- Selection time (if incubation with a selection agent): 9 days

SELECTION AGENT (mutation assays): 6-Thioguanine


NUMBER OF REPLICATIONS: 2


NUMBER OF CELLS EVALUATED: >1.5x10E6


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

A test item is classified as clearly mutagenic if, in any of the experimental conditions examined, all of the following criteria are met:
a. at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b. the increase is dose-related when evaluated with an appropriate trend test,
c. any of the results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).
A test item is classified as clearly non-mutagenic if, in all experimental conditions examined, all of the following criteria are met:
a. none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b. there is no concentration-related increase when evaluated with an appropriate trend test,
c. all results are inside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).
There is no requirement for verification of a clearly positive or negative response. In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations.
In rare cases, even after further investigations, the data set will preclude making a conclusion of positive or negative results, and therefore the test chemical response will be concluded to be equivocal.

Statistics:

A linear regression (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend was judged as significant whenever the p-value (probability value) is below 0.05.
A t-Test was performed using a validated test script of “R”, a language and environment for statistical computing and graphics, to evaluate a significant increase of the mutation frequency at test points exceeding the 95% confidence interval. Again a t-test was judged as significant if the p-value (probability value) is below 0.05.
However, both, biological and statistical significance were considered together.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not effected (pH 7.43 in the solvent control versus pH 7.39 at 10000.0 µg/mL)
- Effects of osmolality: No relevant increase (291 mOsm in the solvent control versus 309 mOsm at 10000 µg/mL)
- Precipitation: determined at 312.5, 625.0, 1250.0 µg/mL without metabolic activation, and at 156.3, 312.5, 625.0 µg/mL with metabolic activation
- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES:
The pre-experiment was performed in the presence and absence (4 h treatment) of metabolic activation. Test item concentrations between 78.1 µg/mL and 10000 µg/mL were used with respect to the current OECD guideline 476, adopted 29 July 2016, regarding the purity of the test item (approx. 20%).
In the pre-experiment no cytotoxicity was observed up to the highest concentration following 4 hours of treatment with and without metabolic activation.
The test medium was checked for precipitation or phase separation at the beginning and at the end of treatment (4 hours) prior to removal to the test item. At the beginning of treatment precipitation was observed at 2500 µg/ml and above with and without metabolic activation. At the end of the 4 hours treatment precipitation occurred at 312.5 µg/mL and above with metabolic activation and at 625 µg/ml without metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA: Complies

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No relevant cytotoxic effects indicated by a relative adjusted cloning efficiency I below 50% occurred up to the maximum evaluated concentration with and without metabolic activation.

Summary Table

	conc. µg/mL	S9 mix	relative CE I	relative cell density	rel. adj. CE I	mutant colonies per 10^6 cells	95% control limit
Culture I
Solvent control (water)		-	100.0	100.0	100.0	20.7	1.7 - 30.2
Positive control (EMS)	300.0	-	90.3	96.5	87.1	253.0	1.7 - 30.2
Test Item	39.1	-	101.7	100.1	101.9	19.7	1.7 - 30.2
Test Item	78.1	-	101.0	105.0	106.0	25.3	1.7 - 30.2
Test Item	156.3	-	97.6	88.7	86.6	29.1	1.7 - 30.2
Test Item	312.5 (p)	-	97.5	103.1	100.6	32.7	1.7 - 30.2
Test Item	625.0 (p)	-	95.8	88.1	84.3	19.2	1.7 - 30.2
Test Item	1250.0 (p)	-	101.9	181.1	184.6	not continued #
Solvent control (water)		+	100.0	100.0	100.0	22.7	2.0 - 29.4
Positive control (DMBA)	2.3	+	98.0	64.5	63.2	131.1	2.0 - 29.4
Test Item	19.5	+	92.3	105.5	97.4	21.8	2.0 - 29.4
Test Item	39.1	+	98.1	77.6	76.1	27.9	2.0 - 29.4
Test Item	78.1	+	93.4	69.7	65.1	14.3	2.0 - 29.4
Test Item	156.3 (p)	+	94.0	55.0	51.6	22.3	2.0 - 29.4
Test Item	312.5 (p)	+	99.8	50.4	50.3	23.5	2.0 - 29.4
Test Item	625.0  (p)	+	92.0	81.7	75.1	not continued #

	conc. µg/mL	S9 mix	relative CE I	relative cell density	rel. adj. CE I	mutant colonies per 10^6cells	95% control limit
Culture II
Solvent control (DMSO)		-	100.0	100.0	100.0	19.2	1.7 - 30.2
Positive control (EMS)	300.0	-	104.9	102.9	107.9	329.3	1.7 - 30.2
Test Item	39.1	-	107.3	84.4	90.6	13.5	1.7 - 30.2
Test Item	78.1	-	104.9	67.8	71.1	30.0	1.7 - 30.2
Test Item	156.3	-	109.8	55.7	61.2	15.6	1.7 - 30.2
Test Item	312.5 (p)	-	100.4	79.4	79.7	26.4	1.7 - 30.2
Test Item	625.0 (p)	-	101.0	94.4	95.3	26.9	1.7 - 30.2
Test Item	1250.0 (p)	-	96.6	105.9	102.3	not continued #
Solvent control (DMSO)		+	100.0	100.0	100.0	18.4	2.0 - 29.4
Positive control (DMBA)	2.3	+	97.4	35.1	34.2	117.3	2.0 - 29.4
Test Item	19.5	+	91.0	96.2	87.5	13.5	2.0 - 29.4
Test Item	39.1	+	94.3	103.2	97.3	21.9	2.0 - 29.4
Test Item	78.1	+	90.0	97.6	87.8	28.5	2.0 - 29.4
Test Item	156.3 (p)	+	99.6	117.9	117.5	30.2	2.0 - 29.4
Test Item	312.5 (p)	+	95.3	75.4	71.9	21.8	2.0 - 29.4
Test Item	625.0 (p)	+	102.3	140.0	143.2	not continued #

CE = Cloning efficiency

P = precipitation visible to the unaided eye at the end of treatment

#    culture was not continued to avoid analysis of too many precipitating concentrations

Conclusions:

No relevant increases in mutation frequencies were observed after test substance treatment up to the highest required concentraton in the absence and presence of metabolic activation in a gene mutation assay at the hprt locus using V79 cells.

Executive summary:

A gene mutation test at the HPRT locus was performed in Chinese hamster lung cells (V79) up to the highest requested concentration showing precipitation of the substance at top concentrations of 312.5 µg/mL in the presence of S9 mix and 625 µg/mL in the absence of S9 mix. No relevant cytotoxic effects indicated by a relative adjusted cloning efficiency below 50% occurred up to the maximum evaluated concentration with and without metabolic activation.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

Under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells and the substance Leuco Sulphur Brown 96 is considered to be non-mutagenic.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

11-Jun-2012 to 25-Jun-2012

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

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Experiment 1
Preliminary test (without and with S9) TA100 and WP2uvrA: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate
Main study: TA1535, TA1537 and TA98:
Without and with S9-mix: 33, 100, 333, 1000 and 3330 µg/plate
Experiment 2:
Without and with S9-mix: 3, 100, 333, 1000 and 3330 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:
A homogeneous suspension could be obtained in DMSO and DMSO is accepted and approved by authorities and international guidelines

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene: with S9, in DMSO for all tester strains

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive if:
a) A two-fold (TA100) or more or a three-fold (TA1535, TA1537, TA98, WP2uvrA) or more increase above solvent control in the mean number of revertant colonies is observed in the test substance group.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Except in DRF studie, toxicity observed in tester strain TA100

Vehicle 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:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Except in DRF studie, toxicity observed

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation was observed at dose levels of 3330 µg/plate and above in the first experiment and at 1000 µg/plate and above in the second experiment

RANGE-FINDING/SCREENING STUDIES:
- In tester strain TA100, toxicity was observed at dose levels of 3330 and 5000 μg/plate in the absence and presence of S9-mix. In tester strain WP2uvrA, toxicity was observed at the dose level of 5000 μg/plate in the absence and presence of S9-mix.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- No toxicity was observed up to and including the top dose of 3330 µg/plate

Conclusions:

In a bacterial reverse mutation assay in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli strain WP2 uvr no mutagenicity was observed up to the highest required test item concentration in the absence and presence of metabolic activation.

Executive summary:

A bacterial reverse mutation test (Ames test) was performed in Salmonelly typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli strain WP2 uvrA. No toxicity was observed after test item treatment. Evaluation of plates up to precipitating concentration ranges of 3300 µg/plate did not reveal any increases in revertant colonies (His+ or Trp+) in either of the bacterial strains in the absence and presence of metabolic activation. These results were confirmed in an independently repeated experiment.

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

In conclusion, C.I. Leuco Sulphur Brown 96 is considered to be non-mutagenic in the Ames test under the test conditions described.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames test:

A bacterial reverse mutation test (Ames test) was performed in Salmonelly typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli strain WP2 uvrA. No toxicity was observed after test item treatment. Evaluation of plates up to precipitating concentration ranges of 3300 µg/plate did not reveal any increases in revertant colonies (His+ or Trp+) in either of the bacterial strains in the absence and presence of metabolic activation. These results were confirmed in an independently repeated experiment.

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

In conclusion, C.I. Leuco Sulphur Brown 96 is considered to be non-mutagenic in the Ames test under the test conditions described.

Chromosomal aberration test:

In a chromosomal aberration assay using peripheral human lymphocytes with and without a metabolic activation system C.I. Leuco Sulphur Brown 96 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently repeated experiments up to a precipitating concentration range with a top dose of 1000 µg/mL. No cytotoxicity was observed after treatment with the substance.

The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system functioned properly. 

No effects of C.I. Leuco Sulphur Brown 96 on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix.

Therefore it can be concluded that C.I. Leuco Sulphur Brown 96 does not disturb mitotic processes and cell cycle progression and does not induce structural and numerical chromosome aberrations under the experimental conditions described.

HPRT test:

A gene mutation test at the HPRT locus was performed in Chinese hamster lung cells (V79) up to the highest requested concentration showing precipitation of the substance at top concentrations of 312.5 µg/mL in the presence of S9 mix and 625 µg/mL in the absence of S9 mix. No relevant cytotoxic effects indicated by a relative adjusted cloning efficiency below 50% occurred up to the maximum evaluated concentration with and without metabolic activation.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

Under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells and the substance Leuco Sulphur Brown 96 is considered to be non-mutagenic.

Justification for classification or non-classification

Classification, Labelling, 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 considered to be classified for genetic toxicity under Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.