Reaction product of 3,9-dibromobenzanthrone condensed with 2 equivalents of 1-aminoanthraquinone, subsequently further condensed under oxidative conditions

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in vitro bacterial reverse mutation (Ames) assay

In the first Ames study, the test substance was slightly mutagenic in this test system when tested without activation, however, the metabolites formed by metabolic activation, revealed a clear-cut mutagenic effect.

In the second Ames study, the test substance induced reproducible and dose related increases in the number of revertant colonies with TA1537 and TA100 tester strains in the absence of S9 metabolism, and with TA1537 and TA98 tester strains in its presence.

Genetic toxicity in vitro mammalian cell gene mutation assay

In the in vitro mammalian cell gene mutation study in V79 Chinese hamster, the test substance and its metabolites did not show any mutagenic potential.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 20, 1995 to October 24, 1995

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)

Version / remarks:

OECD (April 4, 1984)Genetic Toxicology: In vitro Mammalian Cell Gene Mutation Tests.OECD Guideline for Testing of Chemicals 476.

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

EEC Directive 87/302, Annex (November 18, 1987)Part B; Mutagenicity testing and screening for carcinogenicity; In vitro mammalian cell gene mutation test.Official Journal of the European Communities, No L 133, Vol. 31, 61-63, May 30, 1988

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5300 (Detection of Gene Mutations in Somatic Cells in Culture)

Version / remarks:

EPA (May 20, 1987)Detection of gene mutations in somatic cells in culture. Environmental Protection Agency Health Effects Testing Guidelines52 FR 19072 (Corn 52 FR 26150, July 13, 1987); 798.5300

Deviations:

not specified

GLP compliance:

yes

Type of assay:

other: forward mutation system in mammalian cells

Target gene:

6-TG

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

V79 Chinese hamster cells were originally derived from embryonic lung tissue. The cells were cultured in Ham's F10 medium supplemented with 10% pre-tested foetal calf serum, 100 U/ml penicillin and 100 ug/ml streptomycin in tissue culture (plastic) flasks. The humidity in the incubator was adjusted to >85% rH, the air was enriched to 5 ± 2.0 Vol% CO2 and the temperature was 37±1°C.Twice per week the growth medium was replaced by fresh one.The laboratory cultures were passaged weekly in low number (about 5e4 cells per 175 cm2) to keep the level of spontaneous mutants low and to prevent the cells of reaching a stationary phase of cell growth. Large stocks of the V79 cell line have been stored in liquid nitrogen allowing the repeated use of the same cell culture batch in experiments. Consequently, the parameters of the experiments remain similar because of the reproducible characteristics of the cells. The frozen cell suspension contains 10% dimethylsulfoxide (DMSO). All stock cells were cultured in cleansing medium for three days to purge the cultures of existing hprt" mutants. Cleansing medium was growth medium supplemented with 3 uM aminopterin. The cells have a stable karyotype with a modal chromosome number of 22±1. All stock cells were checked for mycoplasma contamination, using the Hoechst-Dye staining method or the 6-MPDR method, before being frozen. Thawed stock culture cells are kept not longer than for twelve passages (three months) in culture.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction

Test concentrations with justification for top dose:

Cytotoxicity test
Range with metabolic activation: 2.44 to 5000.0 ug/ml
Range without metabolic activation: 2.44 to 5000.0 ug/ml
Mutagenicity test
Original experiment: Range with metabolic activation: 185.19 to 5000.0 ug/ml
Range without metabolic activation: 185.19 to 5000.0 ug/ml
Confirmatory experiment:Range with metabolic activation: 625.0 to 5000.0 ug/ml
Range without metabolic activation: 625.0 to 5000.0 ug/ml
A preliminary range finding test was run assessing cytotoxicity. FAT 45019/E was tested at concentrations up to 5000.0 ug/ml. In the part with metabolic activation no cytotoxic effect could be seen.
Without metabolic activation treatment with FAT 45019/E revealed an acute inhibition of growth of 59.5 and 59.6% at the two highest concentrations. Accordingly, 5000.0 ug/ml with and without metabolic activation was chosen as highest concentration for the first mutagenicity assay.

Vehicle / solvent:

Dimethylsulfoxide (DMSO).FAT 45019/E proved to be insoluble in all common vehicles. Therefore, FAT 45019/E had to be applied as a suspension. DMSO was chosen as vehicle.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

ethylmethanesulphonate

Details on test system and experimental conditions:

Preliminary cytotoxicity test
A cytotoxicity test was performed on V79 cells as a preliminary test to determine the highest concentration of the test substance to be applied in the mutagenicity assay. For each concentration and the untreated controls, 2.5e5V79 cells were seeded in 5 ml growth medium into a 25 cm2 tissue culture flask and incubated overnight. The cultures were exposed to the test substance for five hours in the presence and for 21 hours in the absence of a metabolic activation system. In the two parts of the experiment, 12 concentrations of the test substance and two vehicle (DMSO) controls were tested.The highest concentration was determined in a preliminary solubility test. Lower concentrations were prepared by serial dilution by a factor of 0.5. The treatment was terminated by washing the cultures with phosphate buffered saline (PBS). Compound-induced cytotoxicity was estimated by cloning efficiency immediately after treatment. The cultures were counted and diluted so that 100 cells were seeded per 9.6 cm2 in 3 ml of growth medium. After seven to eight days of growth the cultures were fixed and stained with Giemsa and the surviving colonies determined with the aid of an electronic colony counter (Artek Counter®, Fisher Scientific) or by the naked eye. The sensitivity of the colony counter was adjusted to detect clones of about twenty or more cells. The concentration to be selected as the highest for the mutagenicity assay was the one causing about 50-90% reduction of viable cells in comparison with the mean of the two negative controls or corresponds to the substance's solubility limit (precipitates in the culture).
Mutagenicity test
Depending on the toxicity of the test compound 2.5-5.0e6 cells of passage 24 (original experiment) and passage 24 (confirmatory experiment) were plated in 30 ml growth medium into 175 cm2 flasks and incubated overnight. The growth medium was replaced for five hours by 27 ml treatment medium and 3.0 ml S9 activation mixture, or for 21 hours by 30 ml treatment medium alone.In each assay, cultures were treated in duplicate with four test chemical concentrations, a positive and a negative (DMSO) control. In the non-activated part of the experiment, the positive control was the ultimate mutagen Ethylmethansulphonate (EMS) at a concentration of 0.3 ul/ml. In the part with metabolic activation the positive control was the promutagen N-Nitrosodimethylamine (DMN) at a concentration of 1.0 μl/ml.The treatment was terminated by washing the cell layer extensively with PBS. After washing, the cells were suspended by trypsinisation, pelleted, resuspended in fresh growth medium and counted with a haemocytometer or electronic coulter counter (Coulter Counter®, Model ZM), diluted with fresh growth medium and replated into flasks at 2xl06 cells. The cultures were incubated at 37°C for seven to eight days during which the cells could recover and divide to express the mutant phenotype.The cultures were subcultered after the second or third day transferring 2e6 cells to a fresh flask to maintain exponential growth during the expression phase.In parallel cytotoxicity of the compound was estimated from the cloning efficiency immediately after treatment. The counted cell suspension of each concentration level was further diluted so that 100 cells were seeded per 9.6 cm2 in 2.5 ml of growth medium and incubated at 37°C. The number of colonies which developed within seven to eight days in these cultures reflected the viability at the end of the treatment (survival values).At the end of the expression period the cultures were trypsinised, pelleted, resuspended in fresh growth medium and counted with a haemocytometer or electronic coulter counter (Coulter Counter®, Model ZM). The cell suspension of each culture was diluted with fresh growth medium and an aliquot replated into four flasks (75 cm2 growth area) each containing 2e6 cells for the mutant selection. The high-density cultures were subjected to the mutant selection procedure by supplementing the growth medium with 8 μg/ml 6-thioguanine (6-TG). Only cells mutated at the hprt locus could survive the 6-thioguanine treatment. The number of colonies formed in these flasks during the following days reflected the overall number of mutations induced by the treatment with the test substance or the mutagen (positive control). After seven to eight days incubation at 37°C, the cultures were fixed and stained with Giemsa. The mutant clones were counted with the naked eye.
In parallel the viability at the end of the expression period was estimated from the cloning efficiency.The remaining cell suspensions from the various expression cultures were further diluted such that 100 cells were seeded per 9.6 cm2 in 2.5 ml of growth medium and were incubated at 37°C. The number of colonies which developed within these low-density cultures reflected the viability at the end of the expression period (viability values).
Assay acceptance criteria
The results of the experiments should not be influenced by a technical error, contamination or a recognized artifact.From each experiment, at least three concentrations of the test substance, one positive and one solvent control should be evaluated.The mutant frequency of the solvent controls (spontaneous mutant frequency) should not exceed 35e6.
The positive control should fulfil the criteria for a mutagenic substance.The highest concentration of the test substance applied in the mutagenicity test should either reduce the viable cells by about 50-90% or correspond to the test substance's solubility limit (precipitates in the culture). In case of non-toxic freely soluble compounds the highest tested concentration will be 5 mg/ml. In special cases the highest concentration can be determined by the sponsor.

Rationale for test conditions:

The test system allows the detection of base-pair substitutions, frameshift mutations and deletions induced by the test substance or by its metabolites. Mutagenic effects are manifested by the appearance of cells resistant to 6-TG and can be quantified by comparison of the numbers of 6-TG resistant colonies in the treated and control cultures. To ensure that any mutagenic effect of metabolites of the test substance found in mammals is also detected, an experiment is performed, in which the metabolic turnover of the test material is simulated in vitro by the addition of an activation mixture to the cell cultures containing rat-liver post mitochondrial supernatant (S9 fraction) and cofactors.

Evaluation criteria:

Assay evaluation criteria
All mutant frequencies are normalized to a virtual cloning efficiency of 100% at the end of the expression period. If the cloning efficiency of the viability cultures is lower than 15%, the corresponding mutant frequency is usually not calculated, owing to the high statistical insignificance of the result. For every concentration a mean mutant factor, which is defined as the ratio of the mean mutant frequencies of the treated cultures with the mean mutant frequencies of the solvent control cultures, will be calculated.
Criteria for a positive response
The test substance will be considered to be mutagenic if:
The assay is valid (see assay acceptance criteria)
The mutant frequency at one or more concentrations is significantly greater than that of the negative control and the number of normalized mutant clones in the treated and untreated cultures differs by more than 20.There is a significant dose-relationship as indicated by the linear trend analysis.The effects described above are reproducible.

Statistics:

Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Toxicity
In the preliminary toxicity test with and without metabolic activation 12 concentrations of FAT 45019/E were tested. The concentrations selected ranged from 2.44 to 5000.0 μg/ml and separated by 2-fold intervals. In the part with metabolic activation no growth inhibition was seen at any concentration. In the part without metabolic activation FAT 45019/E exerted a growth inhibitory effect of 59.5 and 59.6% at the two highest concentrations.
Accordingly, four concentrations were selected for the original experiment ranging from 185.19 to 5000.0 μg/ml in the presence and absence of metabolic activation.
In the presence and in the absence of metabolic activation no severe toxic effect could be seen at any concentration.
In the confirmatory experiment concentrations ranging from 625.0 to 5000.0 μg/ml were used for both parts. Again, no serious toxicity was seen in the presence and in the absence of metabolic activation at any concentration.
Mutagenicity
The test substance caused the formation of precipitates in the culture medium at all concentrations tested.
In the presence and absence of metabolic activation, no relevant increase in mutant frequency was observed at any concentration level of FAT 45019/E tested in the original or the confirmatory experiment in comparison with the negative control.
The occasional occurrence of statistically significant differences is considered to be purely fortuitous and not related to treatment with the test compound. The respective values were clearly within the historical control range and no concentration dependency could be seen.The positive controls induced a clear increase in mutant frequency.

LEGEND TO TABLES

*	No data
Tx	No data due to high toxicity
nTx	Not toxic
§	One duplicate lost
Ns	Not significant

(All calculations on the following tables were made by a computer using exact values. The calculated value given in tables are rounded to two or three digits).

 

RESULT OF THE CYTOTOXICITY TEST

Experiment with metabolic activation

Treatment	Cell number after treatment (x10E6)	Survival clones after treatment (per well)
Negative control	1.281	84	88	86	82	75	81
Negative control	1.134	76	81	80	84	72	79
FAT 45019/E:
5000.0000 μg/ml	1.715	83	80	75	78	77	83
2500.0000 μg/ml	1.615	67	69	76	70	74	77
1250.0000 μg/ml	1.418	78	74	69	76	68	70
625.0000 μg/ml	1.531	84	71	80	76	72	74
312.0000 μg/ml	1.431	84	81	77	74	90	82
156.2500 μg/ml	1.262	79	79	88	78	81	84
78.1250 μg/ml	1.541	82	77	87	70	74	83
39.0625 μg/ml	1.554	82	77	87	70	74	79
19.5313 μg/ml	1.246	77	74	75	81	80	79
9.7656 μg/ml	1.320	83	88	93	90	93	86
4.8828 μg/ml	1.187	86	90	76	79	85	87
2.4414 μg/ml	1.006	89	84	79	87	90	88
Treatment	Mean of clones	Number of viable cells (x10E6)			Acute cytotoxicity (% of control)
Negative control	82.67	1.06
Negative control	78.67	0.89
FAT 45019/E:
5000.0000 μg/ml	79.33	1.36			nTX
2500.0000 μg/ml	72.17	1.17			nTX
1250.0000 μg/ml	72.50	1.03			nTX
625.0000 μg/ml	76.17	1.17			nTX
312.0000 μg/ml	81.33	1.16			nTX
156.2500 μg/ml	81.50	1.03			nTX
78.1250 μg/ml	80.17	1.24			nTX
39.0625 μg/ml	78.83	1.23			nTX
19.5313 μg/ml	77.67	0.97			0.76
9.7656 μg/ml	88.83	1.17			nTX
4.8828 μg/ml	83.83	1.00			nTX
2.4414 μg/ml	86.17	0.87			11.11

 

RESULTS OF THE CYTOTOXICITY TEST

Experiment without metabolic activation

Treatment	Cell number after treatment (x10E6)	Survival clones after treatment (per well)
Negative control	3.075	84	90	78	88	83	85
Negative control	2.450	91	96	84	89	97	95
FAT 45019/E:
5000.0000 μg/ml	2.221	40	43	47	47	41	48
2500.0000 μg/ml	2.096	42	50	48	46	46	49
1250.0000 μg/ml	1.733	89	80	65	68	70	74
625.0000 μg/ml	2.355	67	71	70	67	66	68
312.0000 μg/ml	1.677	74	75	73	69	79	78
156.2500 μg/ml	1.895	74	80	77	81	71	70
78.1250 μg/ml	1.785	73	76	72	70	78	76
39.0625 μg/ml	2.285	85	88	84	84	90	81
19.5313 μg/ml	2.096	97	93	90	93	88	87
9.7656 μg/ml	1.921	86	89	83	88	85	92
4.8828 μg/ml	2.254	90	81	80	69	93	87
2.4414 μg/ml	1.956	99	93	102	96	99	91
Treatment	Mean of clones	Number of viable cells (x10E6)			Acute cytotoxicity (% of control)
Negative control	84.67	2.60
Negative control	92.00	2.25
FAT 45019/E:
5000.0000 μg/ml	44.33	0.98			59.47
2500.0000 μg/ml	46.83	0.98			59.59
1250.0000 μg/ml	74.33	1.29			46.96
625.0000 μg/ml	68.17	1.61			33.90
312.0000 μg/ml	74.67	1.25			48.45
156.2500 μg/ml	75.50	1.42			41.10
78.1250 μg/ml	74.17	1.32			45.49
39.0625 μg/ml	85.33	1.95			19.70
19.5313 μg/ml	91.33	1.91			21.17
9.7656 μg/ml	87.17	1.67			31.05
4.8828 μg/ml	81.67	1.84			24.22
2.4414 μg/ml	96.67	1.89			22.15

 

SUMMARY OF THE MUTAGENICITY EXPERIMENT

Experiment with metabolic activation (Original Experiment)

Treatment	Mean of viability clones per well	Mean of mutants per flask	Normalized mean of mutants per flask
Negative control	77.58	11.75	15.15
Positive control DMN 1 μl/ml	56.83	131.25	230.94
FAT 45019/E:
5000.0000 μl/ml	72.33	14.38	19.87
1666.6667 μl/ml	76.92	14.75	19.18
555.5556 μl/ml	77.33	16.61	21.50
185.1852 μl/ml	74.67	14.25	19.08
Treatment	Mean mutant frequency (x10E-6)	Mean mutant factor	Significance (P)
Negative control	7.57
Positive control DMN 1 μl/ml	115.47	15.25	P<0.001
FAT 45019/E:
5000.0000 μl/ml	9.94	1.31	Ns
1666.6667 μl/ml	9.59	1.27	Ns
555.5556 μl/ml	10.75	1.42	0.02<P<0.05
185.1852 μl/ml	9.54	1.26	Ns
Linear relation : Ns

 

SUMMARY OF THE MUTAGENICITY EXPERIMENT

Experiment without metabolic activation (Original Experiment)

Treatment	Mean of viability clones per well	Mean of mutants per flask	Normalized mean of mutants per flask
Negative control	78.08	7.63	9.77
Positive control EMS 0.3 μl/ml	44.58	1293.50	2901.31
FAT 45019/E:
5000.0000 μl/ml	74.58	10.13	13.58
1666.6667 μl/ml	79.50	8.38	10.53
555.5556 μl/ml	75.33	10.25	13.61
185.1852 μl/ml	75.33	12.50	16.59
Treatment	Mean mutant frequency (x10E-6)	Mean mutant factor	Significance (P)
Negative control	4.88
Positive control Ems 0.3 μl/ml	1450.65	297.11	P<0.001
FAT 45019/E:
5000.0000 μl/ml	6.79	1.39	Ns
1666.6667 μl/ml	5.27	1.08	Ns
555.5556 μl/ml	6.80	1.39	Ns
185.1852 μl/ml	8.30	1.70	Ns
Linear relation : Ns

 

SUMMARY OF THE MUTAGENICITY EXPERIMENT

Experiment with metabolic activation (Confirmatory Experiment)

Treatment	Mean of viability clones per well	Mean of mutants per flask	Normalized mean of mutants per flask
Negative control	77.42	3.88	5.01
Positive control DMN 1 μl/ml	57.50	139.75	243.04
FAT 45019/E:
5000.0000 μl/ml	76.67	2.63	3.42
2500.0000 μl/ml	78.50	5.88	7.48
1250.0000 μl/ml	83.50	4.88	5.84
625.0000 μl/ml	79.17	7.13	9.00
Treatment	Mean mutant frequency (x10E-6)	Mean mutant factor	Significance (P)
Negative control	2.50
Positive control DMN 1 μl/ml	121.52	48.56	P<0.001
FAT 45019/E:
5000.0000 μl/ml	1.71	0.68	Ns
2500.0000 μl/ml	3.74	1.50	Ns
1250.0000 μl/ml	2.92	1.17	Ns
625.0000 μl/ml	4.50	1.80	0.01<P<0.02
Linear relation : 0.01<P<0.025

 

SUMMARY OF THE MUTAGENICITY EXPERIMENT

Experiment without metabolic activation (Confirmatory Experiment)

Treatment	Mean of viability clones per well	Mean of mutants per flask	Normalized mean of mutants per flask
Negative control	85.17	2.88	3.38
Positive control EMS 0.3 μl/ml	48.25	1264.75	2621.24
FAT 45019/E:
5000.0000 μl/ml	83.25	4.25	5.11
2500.0000 μl/ml	83.25	3.75	4.50
1250.0000 μl/ml	88.17	5.63	6.38
625.0000 μl/ml	86.67	6.13	7.07
Treatment	Mean mutant frequency (x10E-6)	Mean mutant factor	Significance (P)
Negative control	1.69
Positive control EMS 0.3 μl/ml	1310.62	776.50	P<0.001
FAT 45019/E:
5000.0000 μl/ml	2.55	1.51	Ns
2500.0000 μl/ml	2.25	1.33	Ns
1250.0000 μl/ml	3.19	1.89	Ns
625.0000 μl/ml	3.53	2.09	Ns
Linear relation : Ns

 

Conclusions:

Based on the results of two independently performed experiments and under the given experimental conditions, it is concluded that FAT 45019/E and its metabolites did not show any mutagenic activity in this forward mutation system.

Executive summary:

FAT 45019/E, >90% purity, Ident-Nr. 5546 was tested for mutagenic effects on V79 Chinese hamster cells in vitro. The test substance was suspended in DMSO. The cells were treated in the experiments with metabolic activation for 5 hours and in the experiments without metabolic activation for 21 hours. The results of each experiment were confirmed in a second and independent experiment (confirmatory experiment).

 

This test is in agreement with:

OECD (April 4, 1984); Genetic Toxicology: In vitro Mammalian Cell Gene Mutation Tests. OECD Guideline for Testing of Chemicals 476.

EPA (May 20, 1987); Detection of gene mutations in somatic cells in culture. Environmental Protection Agency Health Effects Testing Guidelines; 52 FR 19072 (Corn 52 FR 26150, July 13, 1987); 798.5300

EEC Directive 87/302, Annex (November 18, 1987); Part B; Mutagenicity testing and screening for carcinogenicity; In vitro mammalian cell gene mutation test. Official Journal of the European Communities, No L 133, Vol. 31, 61-63, May 30, 1988

 

Cytotoxicity test

A preliminary range finding test was run assessing cytotoxicity. FAT 45019/E was tested at concentrations up to 5000.0 μg/ml. In the part with metabolic activation no cytotoxic effect could be seen. Without metabolic activation treatment with FAT 45019/E revealed an acute inhibition of growth of 59.5 and 59.6% at the two highest concentrations. Accordingly, 5000.0 μg/ml with and without metabolic activation was chosen as highest concentration for the first mutagenicity assay.

 

Mutagenicity test with metabolic activation

The original experiment was performed at the following concentrations: 185.19, 555.56, 1666.67 and 5000.0 μg/ml. In the confirmatory experiment the concentrations applied were 625.0, 1250.0, 2500.0 and 5000.0 μg/ml. Both experiments revealed no relevant cytotoxic effects. N-Nitrosodimethylamine (DMN, 1.0 μl/ml) was used as positive control.

In both experiments comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance revealed no relevant increase of the mutant frequencies as determined by the screening with 6-Thioguanine (6-TG).

 

Mutagenicity test without metabolic activation

The original experiment was performed at the following concentrations: 185.19, 555.56, 1666.67 and 5000.0 μg/ml. In the confirmatory experiment the concentrations applied were 625.0, 1250.0, 2500.0 and 5000.0 μg/ml. No severe toxicity was observed in the two experiments. Ethylmethanesulfonate (EMS, 0.3 μl/ml) was used as positive control.

In both experiments comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance revealed no relevant increase of the mutant frequencies as determined by the screening with 6-TG.

 

Based on the results of two independently performed experiments and under the given experimental conditions, it is concluded that FAT 45019/E and its metabolites did not show any mutagenic activity in this forward mutation system.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

October 14, 1992 to February 19, 1993.

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:

OCED GUIDELINES FOR TEST OF CHEMICALS, No. 471, Genetic Toxicology: Salmonella typhimurium, Reverse Mutation Assay, May 26, 1983

Deviations:

yes

Remarks:

In deviation to the OECD guideline a statistical analysis was not performed

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OFFICIAL JOURNAL OF THE EUROPEAN COMMINITIES, No. L 251/143-145 Part B, 14: Salmonella typhimurium – Reverse Mutation Assay, September 19, 1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Version / remarks:

EPA HEALTH EFFECTS TESTING GUIDELINES, 40 CFR 789, corrected at 52 FR 26150, § 798.5265, Salmonella typhimurium, Reverse Mutation Assay, July 13, 1987.

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

The histidine-auxotrophic strains of Salmonella typhimurium (TA 98, TA 100, TA 1535, TA 1537 and TA 1538) were obtained from Prof. B. Ames, Berkeley, CA., USA.

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 1538

Details on mammalian cell type (if applicable):

The histidine-auxotrophic strains of Salmonella typhimurium (TA 98, TA 100, TA 1535, TA 1537 and TA 1538) were obtained from Prof. B. Ames, Berkeley, CA., USA.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

Range in the cytotoxicity test 20.6 – 5000 μg/plate; Range in the mutagenicity test 61.7 – 5000 μg/plate
The highest concentration applied was 5000 μg/plate (because of lack of toxicity in the range finding test) and the four lower concentrations were each decreased by a factor of 3.

Vehicle / solvent:

Vehicle: Dimethylsulfoxide (suspension)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

cyclophosphamide

other: 2-aminoanthracene

Details on test system and experimental conditions:

Control of the genotype of the strains
The characteristics of the strains were checked monthly. Histidine-auxotrophy of the strains was demonstrated by the requirement for l-histidine. The presence of the rfa character was assayed by the sensitivity for crystal-violet. The deletion of the uvrB gene was demonstrated by the sensitivity for UV-light. The Salmonella strains containing the R-factor (TA 98 and TA 100) were additionally checked for ampicillin resistance. Furthermore, all strains were checked for their characteristic reversion properties with known mutagens (positive control). Preparation of the metabolic activation mixtureRat-liver microsomal fraction S9 was prepared in advance from male RAI rats (Tif: RAIf[SPF]), reared at the animal farm of CIBA-GEIGY, Sisseln, Switzerland. The animals (150-250 g) were treated with Aroclor 1254 (500 mg/kg i.p.) 5 days prior to sacrifice. The livers were homogenized with 3 volumes of 150 mM KCI and the 9000x g supernatant (S9) was stored at approximately -80°C for no longer than one year. The protein content of the S9 fraction was 24.5 mg/ml. Setting up of the test plates0.1 ml of the overnight cultures were mixed with 2 ml of top agar, either 0.5 ml of 100 mM sodium phosphate buffer (experiments without activation) or 0.5 ml of the activation mixture (experiments with activation) and 0.1 ml of a solution of the test substance, the substance for the positive control or the solvent for the negative control and poured on minimal agar in Petri dishes. Each Petri dish contained about 20 ml of minimal agar (1.5% agar supplemented with 2% salts of the Vogel-Bonner Medium E and 2% glucose). The top agar was composed of 0.6% agar and 0.6% NaCl. It was supplemented with 10% of 0.5 mM l-histidine and 0.5 mM (+)biotin dissolved in water. Preliminary Toxicity/Range-Finding TestA toxicity test (check for reduction in the number of revertant colonies) was carried out with strain TA 100 without and with microsomal activation at six concentrations of the test substance and one negative control according to Standard Operating Procedures of Genetic Toxicity. The highest concentration applied was 5000 μg/plate. The five lower concentrations decreased by a factor of 3. The plates were inverted and incubated for about 48 hours at 37 ± 1.5°C in darkness. Thereafter, they were evaluated by counting the colonies and determining the background lawn. One plate per test substance concentration, as well as each negative control was used. Mutagenicity testThe mutagenicity test was performed with strains TA 98, AT 100, TA 1535, TA 1537 and TA 1538 without and with microsomal activation according to Standard Operating Procedures of Genetic Toxicity. Each of the five concentrations of the test substance, a negative and a positive control were tested, using three plates per test substance concentration as well as each positive and negative control with each tester strain. The highest concentration applied was 5000 μg/plate (because of lack of toxicity in the range finding test) and the four lower concentrations were each decreased by a factor of 3. The plates were inverted and incubated for about 48 hours at 37 ± 1.5°C in darkness.Thereafter, they were evaluated by counting the number of colonies and determining the background lawn.Colony counting and scoring of the platesColonies were counted electronically with an Artex counter. The results were sent on line to a computer. They were checked on a random basis by the operator. Observations indicating precipitates of the test substance in the top agar or a reduced or absent bacterial background lawn were registered additionally. Means and standard deviations for all mutagenicity assays were calculated by a previously validated computer program.

Rationale for test conditions:

The experiments were performed to detect any properties of the test material or its metabolites to induce gene mutations in histidine-requiring strains of Salmonella typhimurium. When salmonella strains are exposed to a mutagen, some of the bacteria in the treated population, through chemical interaction with the compound, undergo genetic changes which cause them to revert to a non-histidine-requiring state and thus to grow in the absence of exogenous histidine. Mutagenic effects of the test substance are demonstrable on comparison of the number of bacteria in the treated and control cultures that have undergone reverse-mutation to histidine prototrophism. Different tester strains are used because of differing sensitivities to known mutagens.

Evaluation criteria:

Assay acceptance criteria
A test is considered acceptable if the mean colony counts of the control values of all strains are within the acceptable ranges and if the results of the positive controls meet the criteria for a positive response. In either case the final decision is based on the scientific judgement of the Study Director. Criteria for a positive response
The test substance is considered to be mutagenic in this test system if the following conditions are met:
At least a reproducible meaningful increase of the mean number of revertants per plate above that of the negative control, at any concentration for one or more of the following strains: S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA 1538.
Generally a concentration-related effect should be demonstrable.

Statistics:

In deviation to the OECD guideline a statistical analysis was not performed. At present the use of statistical methods concerning this particular test system is not generally recommended. No appropriate statistical method is available

Key result

Species / strain:

S. typhimurium, other: strains TA 98, TA 100, TA 1537 and TA 1538

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Toxicity test/range finding test
Six concentrations of Cibanon olive S (FAT 45019/E) ranging from 20.6 – 5000 μg/plate were tested with strain S. typhimurium TA 100 to determine the highest concentration to be used in the mutagenicity assay. The experiments were performed with and without microsomal activation. Normal background growth was observed. A concentration dependent increase in the numbers of revertant colonies was observed in the experiment with activation. From the results obtained, the highest concentration suitable for the mutagenicity test was selected to be 5000 μg/plate without and with activation. Mutagenicity test, original experimentIn the experiment performed without microsomal activation with strains TA 98, TA 100, TA 1535 and TA 1538, after treatment with Cibanon olive S (FAT 45019/E) no increase in the incidence of histidine-prototrophic mutants was observed in comparison with the negative control. With strain TA 1537 a slight increase in the number of back-mutants occurred at the concentrations of 185.2 to 1666.7 μg/plate. In the experiment performed with activation, treatment with Cibanon olive S (FAT 45019/E) lead to an increase in the number of revertant colonies with strain TA 100 at the concentration of 555.6 μg/plate and above. With strains TA 98, TA 1537 and TA 1538 this effect was more pronounced and occurred at all concentrations tested. Mutagenicity test, confirmatory experimentIn the experiment performed without microsomal activation, treatment with Cibanon olive S (FAT 45019/E) lead only to a slight increase in the number of back-mutants with strain TA 1537. In the experiment performed with activation, treatment with Cibanon olive S (FAT 45019/E) lead again to an increase in the number of revertant colonies with strain TA 100 at the concentrations of 555.6 μg/plate and above and with strains TA 98, TA 1537 and TA 1538 at all concentrations tested.In the mutagenicity tests a reduction in background growth was occasionally observed at the two highest concentrations. The test substance thus exerted a slight toxic effect on the bacteria at these concentrations.

RESULTS OF THE RANGE FINDING TEST

TA 100, Experiment without metabolic activation

Treatment	Colony counts
Negative control	134
FAT 45019/E:
20.5761 μg/plate 61.7284 μg/plate 185.1852 μg/plate 555.5556 μg/plate 1666.6667 μg/plate 5000.000 μg/plate	139 121 111 113 117 138
Treatment	Remarks	Relative to control
Negative control	--	1.00
FAT 45019/E:
20.5761 μg/plate 61.7284 μg/plate 185.1852 μg/plate 555.5556 μg/plate 1666.6667 μg/plate 5000.0000 μg/plate	-- -- -- -- -- --	1.04 .90 .83 .84 .87 1.03

-- : No remarks

 

RESULTS OF THE RANGE FINDING TEST

TA 100, Experiment with metabolic activation

Treatment	Colony counts
Negative control	126
FAT 45019/E:
20.5761 μg/plate 61.7284 μg/plate 185.1852 μg/plate 555.5556 μg/plate 1666.6667 μg/plate 5000.0000 μg/plate	152 157 228 324 545 558
Treatment	Remarks	Relative to control
Negative control	--	1.00
FAT 45019/E:
20.5761 μg/plate 61.7284 μg/plate 185.1852 μg/plate 555.5556 μg/plate 1666.6667 μg/plate 5000.0000 μg/plate	-- -- -- -- -- --	1.21 1.25 1.81 2.57 4.33 4.43

-- : No remarks

 

SUMMARY OF THE RESULTS (MEAN OF COLONY COUNTS)

Experiment without metabolic activation (Original)

Treatment/Strain	TA 100	TA 1535	TA 1537	TA 1538	TA 98
Negative control	112.7	16.3	6.3	10.3	17.3
FAT 45019/E:
61.7284 μg/plate 185.1852 μg/plate 555.5556 μg/plate 1666.6667 μg/plate 5000.0000 μg/plate	130.0 124.7 121.3 122.3 121.3	12.7 16.7 13.3 11.0 12.3	8.3 14.7 24.3 21.3 9.0	7.0 9.7 10.0 9.7 7.0	13.3 17.7 27.3 22.3 26.3
Positive controls:
Sodium azide 9-aminoacridine 2-nitrofluorene	1608.3 ---- ----	1180.3 ---- ----	---- 2333.0 ----	---- ---- 389.0	---- ---- 1742.7

 

SUMMARY OF THE RESULTS (MEAN OF COLONY COUNTS)

Experiment with metabolic activation (Original)

Treatment/Strain	TA 100	TA 1535	TA 1537	TA 1538	TA 98
Negative control	146.3	11.3	5.7	29.3	44.3
FAT 45019/E:
61.7284 μg/plate 185.1852 μg/plate 555.5556 μg/plate 1666.6667 μg/plate 5000.0000 μg/plate	139.0 157.0 325.3 546.0 384.0	12.0 16.0 19.7 23.7 17.7	11.0 26.7 139.7 174.7 123.7	57.7 100.7 979.0 991.0 1229.0	141.0 263.3 1783.3 2466.3 2473.3
Positive controls:
2-aminoanthracene Cyclophosphamide	2146.3 ----	---- 437.7	244.0 ----	1867.3 ----	2042.3 ----

 

SUMMARY OF THE RESULTS (MEAN OF COLONY COUNTS)

Experiment without metabolic activation (Confirmatory)

Treatment/Strain	TA 100	TA 1535	TA 1537	TA 1538	TA 98
Negative control	90.3	11.0	6.7	10.7	15.0
FAT 45019/E:
61.7284 μg/plate 185.1852 μg/plate 555.5556 μg/plate 1666.6667 μg/plate 5000.0000 μg/plate	85.0 96.7 82.3 95.0 84.3	11.0 9.7 8.3 8.0 7.3	9.7 14.3 14.3 19.0 15.7	8.7 12.0 9.0 6.7 6.0	14.0 15.3 16.7 15.0 14.3
Positive controls:
Sodium azide 9-aminoacridine 2-nitrofluorene	1036.3 ---- ----	880.7 ---- ----	---- 2545.3 ----	---- ---- 312.0	---- ---- 1182.3

 

SUMMARY OF THE RESULTS (MEAN OF COLONY COUNTS)

Experiment with metabolic activation (Confirmatory)

Treatment/Strain	TA 100	TA 1535	TA 1537	TA 1538	TA 98
Negative control	95.7	11.0	9.0	20.0	18.7
FAT 45019/E:
61.7284 μg/plate 185.1852 μg/plate 555.5556 μg/plate 1666.6667 μg/plate 5000.0000 μg/plate	111.0 132.0 231.0 419.0 343.7	10.0 15.3 23.0 27.7 17.3	23.7 55.7 174.3 342.3 334.3	51.3 197.3 918.7 1770.0 1843.7	157.7 572.0 1419.3 2445.7 2588.0
Positive controls:
2-aminoanthracene Cyclophosphamide	1795.3 ----	---- 448.0	306.7 ----	1406.3 ----	2354.3 ----

Conclusions:

Based on the results of the experiments and on standard evaluation criteria, it is concluded that Cibanon olive S (FAT 45019/E) was slightly mutagenic in this test system when tested without metabolic activation. However, the metabolites of Cibanon olive S (FAT 45019/E), formed by metabolic activation, revealed a clear-cut mutagenic effect.

Executive summary:

Purpose: To evaluate the test compound for mutagenic activity in bacterial test systems in the absence and presence of a rat liver S9 activity system.

 

The test procedure is based on:

OECD GUIDELINES FOR TEST OF CHEMICALS, No. 471, Genetic Toxicology: Salmonella typhimurium, Reverse Mutation Assay, May 26, 1983

OFFICIAL JOURNAL OF THE EUROPEAN COMMINITIES, No. L 251/143-145 Part B, 14: Salmonella typhimurium – Reverse Mutation Assay, September 19, 1984

EPA HEALTH EFFECTS TESTING GUIDELINES, 40 CFR 789, corrected at 52 FR 26150, § 798.5265, Salmonella typhimurium, Reverse Mutation Assay, July 13, 1987.

 

Test system

This test system permits the detection of gene mutations induced by the test material or its metabolites in histidine-requiring strains of Salmonella typhimurium.

 

Concentrations tested

The concentration range of Cibanon olive S (FAT 45019/E) to be tested in the mutagenicity test was determined in a preliminary toxicity test. Thus, the substance was tested for mutagenic effects without and with metabolic activation at five concentrations in the range of 61.7 to 5000 μg/plate. IN order to confirm the results, the experiments were repeated in an independent experiment with the same concentrations.

 

Toxicity test/Range finding test

In the experiment with activation Cibanon olive S (FAT 45019/E) led to an increase in the number of revertants at the upper concentrations. This effect did not occur in the experiment without activation. The test material exerted no inhibitory effect on the growth of the bacteria.

 

Mutagenicity test, original experiment

In the original experiment performed with metabolic activation, treatment of strain TA 1537 with Cibanon olive S (FAT 45019/E) in the concentrations of 185.2 to 1666.7 μg/plate led to a slight increase in the incidence of histidine-prototrophic mutants by comparison with the negative control. No effects were observed with the other strains. In the experiment performed with activation, treatment with Cibanon olive S (FAT 45019/E) lead to a concentration dependent increase of revertant growth with strains TA 98, TA 100, TA 1537 and TA 1538.

 

Mutagenicity test, confirmatory experiment

In the confirmatory experiment performed without metabolic activation, again a slight increase in the incidence of histidine-prototrophic mutants by comparison with the negative control occurred with strain TA 1537. In the experiment performed with activation, treatment with Cibanon S (FAT 45019/E) led to an increase of revertant growth with all strains.

 

Conclusion

Based on the results of these experiments and on standard evaluation criteria, it is concluded that Cibanon olive S (FAT 45019/E) was slightly mutagenic in this test system when tested without metabolic activation. However, the metabolites of Cibanon olive S (FAT 45019/E), formed by metabolic activation, revealed a clear-cut mutagenic effect.

Reference 3

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:

04 December 2015-29 January 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial gene mutation assay

Target gene:

The test item Vat Black 25 was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy.

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:

S9 liver homogenate from induced rat ( rat mixed induction)

Test concentrations with justification for top dose:

Main Assay I was performed at the following dose levels:
TA1535, -S9: 500, 250, 125, 62.5, 31.3, 15.6 mcg/plate
TA 1535, +S9: 250, 125, 62.5, 31.3, 15.6, 7.81 mcg/plate
TA1537, +/- S9: 125, 62.5, 31.3, 15.6, 7.81 mcg/plate
WP2 uvrA +/-S9: 500, 250, 125, 62.5, 31.3 mcg/plate
TA98, -S9: 500, 250, 125, 62.5, 31.3, 15.6 mcg/plate
TA98, +S9: 500, 250, 125, 62.5, 31.3 mcg/plate
TA100, +/-S9: 300, 150, 75.0, 37.5, 18.8, 9.38 mcg/plate

Main Assay II was performed at the following dose levels:
TA1537, -S9: 125, 62.5, 31.3, 15.6, 7.81 mcg/plate
TA1537, +S9: 250, 125, 62.5, 31.3, 15.6, 7.81 mcg/plate
TA98, +/-S9: 500, 250, 125, 62.5, 31.3 mcg/plate
TA100, +/-S9: 300, 150, 75.0, 37.5, 18.8, 9.38 mcg/plate

An additional experiment (Main Assay III) was performed using the following dose levels: 250, 125, 62.5, 31.3, 15.6 µg/plate.

Main Assay IV was performed at the following dose levels:
TA1535, -S9: 500, 250, 125, 62.5, 31.3 mcg/plate
TA1535, +S9: 125, 62.5, 31.3, 15.6, 7.81 mcg/plate
TA1537, -S9: 125, 62.5, 31.3, 15.6, 7.81 mcg/plate
TA1537, +S9: 250, 125, 62.5, 31.3, 15.6 mcg/plate
WP2 uvrA, +/-S9: 500, 250, 125, 62.5, 31.3 mcg/plate
TA98, +/-S9: 500, 250, 125, 62.5, 31.3 mcg/plate
TA100, +/-S9: 500, 250, 125, 62.5, 31.3, 15.6 mcg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

other: Methylmethanesulphonate; sodium azide;2 aminoanthracene

Details on test system and experimental conditions:

Toxicity, Main assay I, II and III were perfomed using the plate incorporation method.Main assay IV was perfomed using the pre-incubation method.

Evaluation criteria:

For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.

Statistics:

Doubling rate (Chu et al. 1981).Regression line

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test item induced reproducible and dose related increases in the number of revertant colonies with TA1537 and TA100 tester strains in the absence of S9 metabolism, and with TA1537 and TA98 tester strains in its presence. These increases were greater than twice the concurrent negative control value and so can be considered a clear evidence of mutation induction.

Remarks on result:

other: all strains/cell types tested

Conclusions:

It is concluded that the test item Vat Black 25 induces reverse mutation in Salmonella typhimurium under the reported experimental conditions.

Executive summary:

The test item Vat Black 25 was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy. The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with Phenobarbital and 5,6-Benzoflavone. The test item was used as a suspension in dimethylsulfoxide (DMSO).

Toxicity test: Based on results obtained in a preliminary solubility trial, the test item Vat Black 25 was assayed in the toxicity test at the maximum concentration of 500 µg/plate and at four lower concentrations spaced at approximately half-log intervals: 158, 50.0, 15.8 and 5.00 µg/plate. At the end of the incubation period, no precipitation of the test item was observed with any tester strain, at any dose level, in the absence or presence of S9 metabolism. Toxicity was observed at the highest or two highest dose levels both in the absence and presence of S9 metabolic activation with all tester strains with the exception of WP2 uvrA and TA98 which showed slight toxicity only in the absence of S9 metabolism. Dose related increases in revertant colonies were observed with TA100 tester strain in the absence and presence of S9 metabolism.

Main Assay I: On the basis of toxicity test results, in Main Assay I, using the plate incorporation method, the test item was assayed at the following dose levels:

TA1535: (- S9) 500, 250, 125, 62.5, 31.3 , 15.6 µg/plate.

(+S9) 250, 125, 62.5, 31.3, 15.6, 7.81 µg/plate.

TA1537: (± S9) 125, 62.5, 31.3, 15.6, 7.81 µg/plate.

WP2 uvrA: (± S9) 500, 250, 125, 62.5, 31.3 µg/plate.

TA98: (- S9) 500, 250, 125, 62.5, 31.3, 15.6 µg/plate.

(+S9) 500, 250, 125, 62.5, 31.3 µg/plate.

TA100 : (± S9) 300, 150, 75.0, 37.5, 18.8, 9.38 µg/plate.

Toxicity was observed with TA1537 and TA100 at the highest and two highest dose levels, respectively, in the absence of S9 metabolic activation and with TA1535 at the two highest dose levels in the presence of S9 metabolic activation. Dose related increases in revertant numbers up to 9.5-fold at the highest concentration were observed at all dose levels with TA1537 in the absence of S9 metabolism and at the three highest dose levels with TA98 up to 5.7 fold in the presence of S9 metabolism.

Mutation results obtained in Main Assay I with TA1537, TA98 and TA100 were not coherent with the results obtained in the preliminary toxicity test. An increase in revertant colonies not indicated in the toxicity test was observed, with TA1537 and TA98, while a lower increase was noticed with TA100 tester strain. Based on these results an additional experiment (Main Assay II), using the plate incorporation method, was performed with these tester strains, both in the absence and presence of S9 metabolic activation. The same concentrations were used as in the first assay except in TA1537 with S9, where concentrations of 250,125, 62.5, 31.3, 15.6,7.81 µg/plate were used, because no toxicity was seen in the first assay.

Dose related increases in revertant numbers were observed with TA1537 both in the absence and presence of S9 metabolism (10.5 fold and 5.1-fold respectively) and TA98 in its presence (4.5-fold). Mild increase (2-fold the concurrent negative control) were observed with TA98 and TA100 in the absence of S9 metabolic activation at the highest concentration. In order to clarify inconsistent results obtained with TA1537 tester strain in the presence of S9 metabolic activation, an additional experiment (Main Assay III) was performed with this tester strain, using the plate incorporation method and once again test item treatments yielded a 2-, 2.2- and 3.6-fold dose related increases in revertant numbers in the three highest concentrations. In order to confirm the results observed using the plate incorporation method, an additional experiment, using the pre-incubation method (Main Assay IV) was performed with all tester strains. Dose levels, displayed in the following table, were selected based on toxicity results obtained in all the previous experiments.

TA1535: (- S9) 500, 250, 125, 62.5, 31.3

(+S9) 125, 62.5, 31.3, 15.6, 7.81

TA1537: (- S9) 125, 62.5, 31.3, 15.6, 7.81

(+S9) 250, 125, 62.5, 31.3, 15.6

WP2 uvrA: (± S9) 500, 250, 125, 62.5, 31.3

TA98: (±S9) 500, 250, 125, 62.5, 31.3

TA100: (±S9) 500, 250, 125, 62.5, 31.3, 15.6

Slight toxicity was observed at the highest dose level with TA1535, both in the absence and presence of S9 metabolic activation, and at the two highest dose levels with TA100 in the presence of S9 metabolism. Dose related increases in revertant numbers were observed with TA1537 and TA100 tester strains in the absence of S9 metabolism up to 5- and 2- fold at the highest dose levels, respectively. In the presence of S9, an increase of mutation frequency of 2.45 was observed at the highest concentration with TA1537 whereas TA98 showed increases in revertant number of 3- and 4 fold in the two highest concentrations.

Conclusion: It is concluded that the test item Vat Black 25 induces reverse mutation in some strains of Salmonella typhimurium under the reported experimental conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Genetic toxicity in vivo – Micronucleus test

In the in vivo micronucleus study (conducted as part of the OECD 422 study), the test substance did not induce micronuclei in the polychromatic erythrocytes of treated rats.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 May 2015 to 10 March 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The Sprague Dawley rat was the species and strain of choice because it is accepted by many regulatory authorities and because there is ample experience
and background data on this species and strain.

Sex:

male

Details on test animals or test system and environmental conditions:

A total of 125 Sprague Dawley [Crl:CD(SD)BR] rats (65 males and 60 virgin females), 7 to 8 weeks old and weighing 200 to 225 g for males and 175 to 200 g for females, were ordered from and supplied by Charles River Italia S.p.A., Calco (Lecco), Italy.After arrival, on 21 May 2015, the weight range for each sex was determined (189-216 g for females, 226-250 g for males, slightly outside the range at order) and the animals were temporarily identified within the cage by means of a coloured mark on the tail.A health check was then performed by a veterinarian. An acclimatisation period of at least 14 days was allowed before the start of treatment, during which time the health status of the animals was assessed by thorough observations.The animals were housed in a limited access rodent facility. Animal room controls were set to maintain temperature and relative humidity at 22°C +/- 2°C and 55% +/- 15% respectively; actual conditions were monitored, recorded and the records retained. No relevant deviations from these ranges were recorded during the study. There were approximately 15 to 20 air changes per hour and the rooms were lit by artificial light for 12 hours each day.

Route of administration:

oral: gavage

Vehicle:

The vehicle for the test item was sesame oil.
The vehicle for the positive control item was sterile water for injection.
Test item
The required amount of Vat Black 25 was dissolved/suspended in the vehicle. The formulations were prepared daily (concentrations of 12,5, 50 and 200mg/mL). Concentrations were calculated and expressed in terms of test item as supplied.
Positive Control item
The required amount of positive control item was dissolved in the vehicle. The formulation was prepared on the day of dosing at a concentration of0.2 mg/mL.

Details on exposure:

The test item was administered orally by gavage at a dose volume of 5 mL/kg body weight. Control animals received the vehicle alone at the same dose volume. The dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal.
Analysis was not performed to confirm that the proposed formulation procedure was acceptable and that the stability of the formulation was satisfactory as the test item is neither extractable in hydrophilic nor in lipophilic solvents. The correct concentration preparation was monitored in the weighing record of the test item in each formulation process.
Positive Control
Animals received a single dose approximately 24 hours before sacrifice. Mitomycin-C (positive control) was administered once by intraperitoneal injection at the dose volume of 10 mL/kg body weight. The dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal.

Duration of treatment / exposure:

Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing, through the mating period and thereafter through the day before necropsy (Days 43 and 44 of study). They were treated for a total of 42 or 43 days. Dose volumes were adjusted once per week for each animal according to the last recorded body weight.

Frequency of treatment:

Once a day

Dose / conc.:

62.5 mg/kg bw/day

Dose / conc.:

250 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

Each main group comprised 10 male and 10 female rats (Groups 1 to 4). Two groups (control and high dose levels) included 5 animals per sex which were sacrificed after 2 weeks of recovery (Groups 5 and 6). For genotoxicity endpoint, a satellite control group (Positive Control group) comprised 5 male rats (Group 7).

Control animals:

yes, concurrent vehicle

Positive control(s):

For genotoxicity endpoint, a satellite control group (Positive Control group) comprised 5 male rats (Group 7).The required amount of positive control item was dissolved in the vehicle (sterile water for injection). The formulation was prepared on the day of dosing at a concentration of 0.2 mg/mL. No documents on Mitomycin-C are included in the report. Determination of the stability and concentration of solutions of the positive control item was not undertaken.Mitomycin-C (positive control) was administered once by intraperitoneal injection at the dose volume of 10 mL/kg body weight. The dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal.Positive Control group animals were killed under carbon dioxide asphyxiation.

Tissues and cell types examined:

Bone marrow from one femur of males only.

Details of tissue and slide preparation:

Extraction of bone marrow
Samples of bone marrow were collected approximately 24 and 48 hours after the 2 final treatments, from the same 5 males of the main groups randomly selected for clinical pathology investigation (see section 4.4). Samples of bone marrow were also collected approximately 24 hours after the single treatment from all males of Group 7 (Positive Control group). One femur of each animal was rapidly dissected out and cleaned of surrounding tissue. In order to extract the bone marrow, the bone was cut at the proximal end and irrigated with foetal calf serum using a syringe. The suspension of cells was aspirated, and this procedure was repeated several times.Preparation of the smearsThe suspension thus obtained was centrifuged at 1000 rpm for at least 5 minutes and the supernatant was completely removed. The cells of the sediment were resuspended and transferred onto clean microscope slides as smear preparations. They were air-dried and then fixed with methanol for 10 minutes. Subsequently, slides were stained with haematoxylin and eosin solutions. Finally, the slides were rinsed in distilled water and allowed to dry.Scoring of the slides and data analysisFor each animal, at least three slides were prepared. These slides were randomised and coded by staff not subsequently involved in the scoring.The adequate quality and the sufficient number of cells were evaluated before scoring. Scoring was performed using a microscope and high-power objective. Immature polychromatic erythrocytes (PCEs) stain a pink-purple colour (since they retain basic ribosomal material for approximately 24 hours after enucleation), and can be distinguished from the pink normochromatic erythrocytes (NCE).Erythrocytes lack nuclei, making micronuclei obvious when present; the criteria of Schmid (1976) were used to score micronuclei. At least four thousand polychromatic erythrocytes per animal were scored for the presence of micronuclei. At the same time the number of normochromatic erythrocytes was recorded, as well as the number of micronucleated NCE. The proportion of immature erythrocytes among total erythrocytes gives an indication of the toxicity of the treatment; a reduction in the proportion indicates inhibition of cell division. Finally, the incidence of micronucleated PCE provides an index of induced genetic damage.

Evaluation criteria:

Acceptance criteria
The assay was considered valid if the following criteria were met:
– The incidence of micronucleated PCEs of the vehicle control group fell within the historical negative control range.
– The positive control item results fell within the historical control range and were significantly increased, at statistical analysis, when comparedwith the concurrent negative control.
– 5 males per group were available for slide analysisEvaluation of results
The test item was considered to induce micronuclei if a statistically significant increase in the micronucleus incidence of polychromatic erythrocytes(at p< 0.05) was observed in any treatment group and a dose-effect relationship was demonstrated. Where statistically significant increases in the incidence of micronucleated PCEs were observed, but all results were inside the distribution of negative control values within this laboratory, then historical control data were used to demonstrate that these increases did not have any biological significance.

Statistics:

Only counts from polychromatic cells were subjected to statistical analysis. Using the original observations (and not the micronucleus frequencies per 1000 cells), a modified chi-squared calculation was employed to compare treated and control groups. The degree of heterogeneity within each group was first calculated and where significant it was taken into account in the comparison between groups. If there was no significant within-group heterogeneity, the chi-squared test was used to compare treated groups with the controls. If there was significant within-groups heterogeneity, then that group was compared with the controls using a variance ratio (F) value calculated from the between-group and within-group chi-squared values.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Incidence of micronucleated cells
Following treatment with the test item, a slight increase in the number of micronucleated PCEs over the concurrent negative control was observed for animals from the low and high dose groups; however, the incidences of micronucleated PCEs were comparable to RTC historical control data for negative control animals. A marked increase in the frequency of micronucleated PCEs was observed in the positive control group.Bone marrow cell toxicityThe ratio of mature to immature erythrocytes and the proportion of immature erythrocytes among total erythrocytes were analysed to evaluate the bone marrow cell toxicity. Based on these results, no relevant inhibitory effect on erythropoietic cell division was observed at any dose level.
Analysis of resultsFollowing treatment with the test item, statistically significant increases in the incidence of micronucleated PCEs over the negative control value were observed for animals from the low and high dose levels (p<0.01). A significant dose effect relationship was found after a trend test evaluation (p<0.05). The dose-related and statistically significant increases of micronucleated PCEs could be attributable to the low incidence of micronucleated cells in the vehicle control group, which fell in the lower part of the distribution range of historical control data. In addition, the frequency of micronucleated immature erythrocytes observed at all dose levels was inside the distribution of historical negative control data (95% upper confidence limit = 2.0), therefore the observed increases were not considered biologically meaningful.

 

Dose level (mg/kg/day)	Incidence in micronucleated PCEs			PCE/s(PCEs+NCEs) % Over the mean control value
	Mean	SE	Range
0.00 62.5 250 1000 Mitomycin-C 2.00 mg/kg	0.2 0.9 0.3 0.9 8.2	0.1 0.3 0.1 0.2 1.2	0.0 – 0.5 0.3 – 1.8 0.0 – 0.5 0.5 – 1.5 5.5 – 12.5	100 101 97 96 97

 

SUMMARY OF INCIDENCE OF MICRONUCLEATED CELLS

INCIDENCE OF MICRONUCLEATED CELLS/1000 CELLS
Dose level mg/kg/day	Scored cells		NCE/PCE Ratio	PCE/(PCE+NCE) Ratio	% over the Mean control value	Polychromatic				Normochromatic
	PCE	NCE				Mean	SE	Min	Max	Mean	SE	Min	Max
0.00 62.5 250 1000	20000 20000 20000 20000	22374 21840 23814 24165	1.12 1.09 1.19 1.21	0.47 0.48 0.46 0.45	100 101 97 96	0.2 0.9 0.3 0.9	0.1 0.3 0.1 0.2	0.0 0.2 0.0 0.5	0.5 1.8 0.5 1.5	0.0 0.0 0.0 0.0	0.0 0.0 0.0 0.0	0.0 0.0 0.0 0.0	0.0 0.2 0.0 0.2
Mitomycin C 2.00	20000	23531	1.18	0.46	97	8.2	1.2	5.5	12.5	0.0	0.0	0.0	0.0

NCE/PCE ratio: The ratio of NCE/PCE calculated as the mean of the ratio values for the individual animals.

PCE/(PCE+NCE) ratio: The ratio of PCE/(PCE+NCE) calculated as the ratio of the total PCEs over the total erythrocytes scored

% over the mean control: Percentage of the PCE/(PCE+NCE) ratio of each treated group value over the negative control value

MEAN:    The group mean incidence of micronucleated PCEs and NCEs

SE:          The standard error of the mean incidence

MIN:       Minimum value observed in an individual animal

MAX:      Maximum value observed in an individual animal

 

HISTORICAL CONTROL DATA

INCIDENCES OF MICRONUCLEATED PCEs (%) (1991 – 2015)
Male animals
	Vehicle controls	Positive controls
Mean	0.7	16.4
SD (σn-1)	0.70	8.41
n	165	164
Upper confidence limit (95%)	2.0	NC
Maximum	3.5	47.5
Minimum	0.0	0.5

SD = standard deviation

n = number of animals

NC = not calculated

Conclusions:

Interpretation of results (migrated information): negative
On the basis of the results obtained, it is concluded that Vat Black 25 does not induce micronuclei in the polychromatic erythrocytes of treated rats, under the reported experimental conditions.

Executive summary:

The purpose of this study was to provide information on toxic effects on male and female rats after repeated dosing with the test item, the micronucleus test was included in order to assess the ability of the test item to induce cytogenetic damage in rat bone marrow, as measured by the induction of micronuclei in polychromatic erythrocytes.

 

Experimental procedures were based on the following guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

 

Incidence of micro nucleated cells

Following treatment with the test item, a slight increase in the number of micro nucleated PCEs over the concurrent negative control was observed for animals from the low and high dose groups; however, the incidences of micro nucleated PCEs were comparable to RTC historical control data for negative control animals.

A marked increase in the frequency of micro nucleated PCEs was observed in the positive control group.

 

Bone marrow cell toxicity

The ratio of mature to immature erythrocytes and the proportion of immature erythrocytes among total erythrocytes were analysed to evaluate the bone marrow cell toxicity. Based on these results, no relevant inhibitory effect on erythropoietic cell division was observed at any dose level.

 

Analysis of results

Following treatment with the test item, statistically significant increases in the incidence of micro nucleated PCEs over the negative control value were observed for animals from the low and high dose levels (p<0.01). A significant dose effect relationship was found after a trend test evaluation (p<0.05). The dose-related and statistically significant increases of micro nucleated PCEs could be attributable to the low incidence of micro nucleated cells in the vehicle control group, which fell in the lower part of the distribution range of historical control data. In addition, the frequency of micro nucleated immature erythrocytes observed at all dose levels was inside the distribution of historical negative control data (95% upper confidence limit = 2.0), therefore the observed increases were not considered biologically meaningful.

 

Conclusions

On the basis of the results obtained, it is concluded that Vat Black 25 does not induce micronuclei in the polychromatic erythrocytes of treated rats, under the reported experimental conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genetic toxicity in vitro – Bacterial reverse mutation (Ames) assay

The test item Vat Black 25 was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy. The five tester strains TA 1535, TA 1537, TA 98, TA 100 and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with Phenobarbital and 5,6-Benzoflavone. The test item was used as a suspension in dimethylsulfoxide (DMSO).

 

Toxicity was observed with TA 1537 and TA 100 at the highest and two highest dose levels, respectively, in the absence of S9 metabolic activation and with TA 1535 at the two highest dose levels in the presence of S9 metabolic activation. Dose related increases in revertant numbers up to 9.5-fold at the highest concentration were observed at all dose levels with TA 1537 in the absence of S9 metabolism and at the three highest dose levels with TA 98 up to 5.7 fold in the presence of S9 metabolism.

Mutation results obtained in Main Assay I with TA 1537, TA 98 and TA 100 were not coherent with the results obtained in the preliminary toxicity test. An increase in revertant colonies not indicated in the toxicity test was observed, with TA 1537 and TA 98, while a lower increase was noticed with TA 100 tester strain. Based on these results an additional experiment (Main Assay II), using the plate incorporation method, was performed with these tester strains, both in the absence and presence of S9 metabolic activation. The same concentrations were used as in the first assay except in TA 1537 with S9, where concentrations of 250,125, 62.5, 31.3, 15.6,7.81 µg/plate were used, because no toxicity was seen in the first assay.

Dose related increases in revertant numbers were observed with TA 1537 both in the absence and presence of S9 metabolism (10.5 fold and 5.1-fold respectively) and TA 98 in its presence (4.5-fold). Mild increase (2-fold the concurrent negative control) were observed with TA 98 and TA 100 in the absence of S9 metabolic activation at the highest concentration. In order to clarify inconsistent results obtained with TA 1537 tester strain in the presence of S9 metabolic activation, an additional experiment (Main Assay III) was performed with this tester strain, using the plate incorporation method and once again test item treatments yielded a 2-, 2.2- and 3.6-fold dose related increases in revertant numbers in the three highest concentrations. In order to confirm the results observed using the plate incorporation method, an additional experiment, using the pre-incubation method (Main Assay IV) was performed with all tester strains.

 

Slight toxicity was observed at the highest dose level with TA 1535, both in the absence and presence of S9 metabolic activation, and at the two highest dose levels with TA 100 in the presence of S9 metabolism. Dose related increases in revertant numbers were observed with TA 1537 and TA 100 tester strains in the absence of S9 metabolism up to 5- and 2- fold at the highest dose levels, respectively. In the presence of S9, an increase of mutation frequency of 2.45 was observed at the highest concentration with TA 1537 whereas TA 98 showed increases in revertant number of 3- and 4 fold in the two highest concentrations.

 

Conclusion: It is concluded that the test item Vat Black 25 induces reverse mutation in some strains of Salmonella typhimurium under the reported experimental conditions.

Genetic toxicity in vitro - Bacterial reverse mutation (Ames) Assay

The concentration range of Cibanon olive S (FAT 45019/E) to be tested in the mutagenicity test was determined in a preliminary toxicity test. Thus, the substance was tested for mutagenic effects without and with metabolic activation at five concentrations in the range of 61.7 to 5000 μg/plate. In order to confirm the results, the experiments were repeated in an independent experiment with the same concentrations.

 

Toxicity test/Range finding test; In the experiment with activation Cibanon olive S (FAT 45019/E) led to an increase in the number of revertants at the upper concentrations. This effect did not occur in the experiment without activation. The test material exerted no inhibitory effect on the growth of the bacteria.

 

Mutagenicity test, original experiment: In the original experiment performed with metabolic activation, treatment of strain TA 1537 with Cibanon olive S (FAT 45019/E) in the concentrations of 185.2 to 1666.7 μg/plate led to a slight increase in the incidence of histidine-prototrophic mutants by comparison with the negative control. No effects were observed with the other strains. In the experiment performed with activation, treatment with Cibanon olive S (FAT 45019/E) lead to a concentration dependent increase of revertant growth with strains TA 98, TA 100, TA 1537 and TA 1538.

 

Mutagenicity test, confirmatory experiment: In the confirmatory experiment performed without metabolic activation, again a slight increase in the incidence of histidine-prototrophic mutants by comparison with the negative control occurred with strain TA 1537. In the experiment performed with activation, treatment with Cibanon S (FAT 45019/E) led to an increase of revertant growth with all strains.

 

Conclusion: Based on the results of these experiments and on standard evaluation criteria, it is concluded that Cibanon olive S (FAT 45019/E) was slightly mutagenic in this test system when tested without metabolic activation. However, the metabolites of Cibanon olive S (FAT 45019/E), formed by metabolic activation, revealed a clear-cut mutagenic effect.

 

Genetic toxicity in vitro - Mammalian Cell Gene Mutation Test (V79 cells)

FAT 45019/E, >90% purity, Ident-Nr. 5546 was tested for mutagenic effects on V79 Chinese hamster cells in vitro. The test substance was suspended in DMSO. The cells were treated in the experiments with metabolic activation for 5 hours and in the experiments without metabolic activation for 21 hours. The results of each experiment were confirmed in a second and independent experiment (confirmatory experiment).

 

Cytotoxicity test

A preliminary range finding test was run assessing cytotoxicity. FAT 45019/E was tested at concentrations up to 5000.0 μg/ml. In the part with metabolic activation no cytotoxic effect could be seen. Without metabolic activation treatment with FAT 45019/E revealed an acute inhibition of growth of 59.5 and 59.6% at the two highest concentrations. Accordingly, 5000.0 μg/ml with and without metabolic activation was chosen as highest concentration for the first mutagenicity assay.

 

Mutagenicity test with metabolic activation

The original experiment was performed at the following concentrations: 185.19, 555.56, 1666.67 and 5000.0 μg/ml. In the confirmatory experiment the concentrations applied were 625.0, 1250.0, 2500.0 and 5000.0 μg/ml. Both experiments revealed no relevant cytotoxic effects. N-Nitrosodimethylamine (DMN, 1.0 μl/ml) was used as positive control.

In both experiments comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance revealed no relevant increase of the mutant frequencies as determined by the screening with 6-Thioguanine (6-TG).

 

Mutagenicity test without metabolic activation

The original experiment was performed at the following concentrations: 185.19, 555.56, 1666.67 and 5000.0 μg/ml. In the confirmatory experiment the concentrations applied were 625.0, 1250.0, 2500.0 and 5000.0 μg/ml. No severe toxicity was observed in the two experiments. Ethylmethansulfonate (EMS, 0.3 μl/ml) was used as positive control.

In both experiments comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance revealed no relevant increase of the mutant frequencies as determined by the screening with 6-TG.

 

Based on the results of two independently performed experiments and under the given experimental conditions, it is concluded that FAT 45019/E and its metabolites did not show any mutagenic activity in this forward mutation system.

Genetic toxicity in vivo – Micronucleus test

The purpose of this study was to provide information on toxic effects on male and female rats after repeated dosing with the test item, the micronucleus test was included in order to assess the ability of the test item to induce cytogenetic damage in rat bone marrow, as measured by the induction of micronuclei in polychromatic erythrocytes.

 

Analysis of results

Following treatment with the test item, statistically significant increases in the incidence of micro nucleated PCEs over the negative control value were observed for animals from the low and high dose levels (p<0.01). A significant dose effect relationship was found after a trend test evaluation (p<0.05). The dose-related and statistically significant increases of micro nucleated PCEs could be attributable to the low incidence of micro nucleated cells in the vehicle control group, which fell in the lower part of the distribution range of historical control data. In addition, the frequency of micro nucleated immature erythrocytes observed at all dose levels was inside the distribution of historical negative control data (95% upper confidence limit = 2.0), therefore the observed increases were not considered biologically meaningful.

 

Conclusions

On the basis of the results obtained, it is concluded that Vat Black 25 does not induce micronuclei in the polychromatic erythrocytes of treated rats, under the reported experimental conditions.

Justification for classification or non-classification

Based on the results of these studies, the test substance is not classified as mutagenic in accordance with the CLP Regulation.