Disodium 4-amino-3,6-bis[[4-[(2,4-diaminophenyl)azo]phenyl]azo]-5-hydroxynaphthalene-2,7-disulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD 471) showed positive results in some strains.
The other in vitro test (OECD 476) does not show any mutagenic effect.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Chinese Hamster ceU line V79, clone 65/3, Dr. D. Wild, Freiburg, Germany

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

no data

Test concentrations with justification for top dose:

In the preliminary toxicity test with and without metabolic activation 12 concentrations of the tested substance were tested. The concentrations selected ranged from 0.24 to 500.0 µg/ml and separated by 2-fold intervals.
In the part with metabolic activation the highest concentration produced an acute growth inhibition of 62.70%. In the part without metabolic activationthe substance exerted a complete growth inhibitory effect at the highest concentration of 500.0 µg/ml. The next lower concentrationsof 250.0 and 125.0 µg/ml revealed an acute growth inhibitory effect of 98.46 and 72.56% respectively.

Accordingly, four concentrations were selected for the original experiment ranging from 18.52 to 500.0 µg/ml and from 4.63 to 125.0 µg/ml in the presence and absence of metabolic activation, respectively.
In the part with metabolic activation, the growth inhibition determined after treatment at the highest concentration of 500.0 µg/ml showed a mean value of 6.91%. After expression growth was inhibited by 13.51%.
In the absence of metabolic activation no significant growth inhibitory effect was seen after treatment and expression.
The highest concentration of 500.0 µg/ml tested in the original experiment with activation was determined to be the highest suitable concentration due to solubility limitations in the vehicle. Therefore the same concentration range was tested in the respective confirmatory experiment, although no severe toxiticy was reached. In the part without metabolic activation a concentration range of 9.26 to 250.0 µg/ml was selected for the confirmatory experiment in order to reach a more pronounced toxicity at the highest concentration. In the presence of metabolic activation no significant acute cytotoxicity was obtained at the highest concentration. After expression growth was inhibited by 9.54%.
In the part without activation, the mean growth inhibition at the highest concentration of 250.0 µg/ml was 27.91% in spite of the increased concentration.

Vehicle / solvent:

No data

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

not specified

Evaluation criteria:

Cytotoxicity is determined my measuring the relative cloning efficiency (survival) of the cultures after the treatment period.
Mutant frequency is determined by seeding known numbers of cells in medium containing the selective agent to detect mutant cells, and in medium without selective agent to determine the cloning efficiency (viability).
After the incubation time, colonies are counted. The mutant frequency is derived from the number of mutant colonies in selective medium and the number of colonies in non-selective medium.

Statistics:

No data

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Interpretation of results:
negative without metabolic activation
negative with metabolic activation

Based on the results of the performed experiments and under the given experimental conditions, it is concluded that the tested substance and its metabolites did not show any mutagenic activity in this forward mutation system.

Executive summary:

Direct Black 19 was tested for mutagenicity, in details for the evalutation of properties to induce gene mutations.

The mutagenicity studies were conducted following OECD 476, gene mutation test with Chinese Hamster cell V79.

The studies were performed in the absence and in the presence of metabolic activation. A dose range with different doses was used. The tested substance shows no mutagenic activity.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro cytogenicity study in mammalian cells or in vitro micronucleus study does not need to be conducted because adequate data from an in vivo cytogenicity test are available

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 97

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from rats

Test concentrations with justification for top dose:

Main Test: 10, 100, 1000, 2500, 5000 µg/plate

Vehicle / solvent:

Distilled water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylenediamine (NPD)

Remarks:

TA97 an TA98 without S9 fraction

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-amino-fluorene (2-AFF)

Remarks:

TA97, TA98, TA100 with Fraction S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA100 and TA1535 without Fraction S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

TA1535 with Fraction S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

PREPARATION OF FRACTION S9: Fraction S9 was obtained from male Wistar rats killed with injections of Delor 106 (mix of polychlorinated phenyls).

PREPARATION OF PLATE:
A) plates of the experiment with metabolic activation contain:
- 0,2 µl of fraction S9
- 0,5 ml of tampon containig the co-factors NADP and glucose-6-phosphate

B) plates of the experiment without metabolic activation contain only distilled water

DURATION
- Expression time (cells in growth medium): 48 to 72 hours

Evaluation criteria:

Cytotoxicity: reduced growth rate or thinning of bacterial lawn
A positve result is based on:
- dose-related and reproducible increase in number of revertant colonies
- doubling of spontaneous mutation rate in at least one tester strains either with or without S9

Statistics:

Based on:
- Dunkel V.C., Chu K.C. (1980): Elsevier North-Holland Biomedical Press: The Predictive Value of Short-Term Screening Tests in
Carcinogenicity Evaluation, 231-240.
- Claxton L.D. et al. (1978): Muta-t. Res. 199, 83-91.

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Remarks:

postive in postive in 1° experiment, negative in 2° experiment

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Interpretation of results:
Positive

The results are different for the four strains. In fact the test compound shows:
- no mutagenic effect for strain TA100 and TA1535
- increase of revertant colonies for strain TA97 in the first experiment and no increase in the second experiment.
- an increase of revertant colonies for the strain TA98 with and without metabolic activation.

In conclusion the substance is considered having mutagenic activity.

Executive summary:

Direct Black 19 was tested for mutagenicity with the strains TA97, TA98, TA100 and TA1535 of Salmonella typhimurium.

The mutagenicity studies were conducted in the standard plate test (Ames Test).

The studies were performed in the absence and in the presence of a metabolizing system derived from rat liver homogenate.

A dose range different doses from 10 µg/plate to 5000 µg/plate was used.

Control plates without mutagen showed that the number of spontaneous revertant colonies was similar to that described in the literature. All the positive control compounds gave the expected increase in the number of revertant colonies.

The test compound proved to be toxic to the bacterial strains and 5000 µg/plate was chosen as top dose level for the mutagenicity study.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

The in vivo available studies does not show any mutagenic effect.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Mollegard Deutshland
- Number: 85 (50 male and 35 female)
- Age at study initiation: between 6 and 8 weeks
- Weight at study initiation: 27-48 g for male, 23-37 g for female
- Housing: mouse were housed in Macrolon Type 2 cages in optimal hygienic conditions.
- Diet: Granular Altromin N 1326
- Water: municipal water ad libitum
- Acclimation period:at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 55 ± 10 %
- Air changes (per hr): 10 times/h
- Photoperiod: 12 h light / 12 h dark

Route of administration:

oral: gavage

Vehicle:

Acqueous solution of ultra - amylopectin (0.7%) with the addiction of two drops of Tween 80 (polysorbate) per 10 ml of solution.

Details on exposure:

single oral administration for the tested item
single oral administration for the negative control
single intraperitoneal application for the positive control

Duration of treatment / exposure:

one single application

Frequency of treatment:

single oral administration for the tested item
single oral administration for the negative control
single intraperitoneal application for the positive control

Post exposure period:

24 h and 48 h

Remarks:

Doses / Concentrations:
1600 mg/kg
Basis:
nominal in water

Remarks:

Doses / Concentrations:
800 mg/kg
Basis:
nominal in water

Remarks:

Doses / Concentrations:
400 mg/kg
Basis:
nominal in water

Remarks:

Doses / Concentrations:
1200 mg/kg
Basis:
nominal in water

No. of animals per sex per dose:

Dose 1600 mg/kg: Observation time: 48 h, 5 male and 5 female
Dose 1600 mg/kg: Observation time: 24 h, 5 male and 5 female
Dose 800 mg/kg: Observation time: 24 h, 5 male and 5 female
Dose 400 mg/kg: Observation time: 24 h, 5 male and 5 female
Dose 1200 mg/kg: Observation time: 24 h, 5 male and 5 female

Positive control: Dose 80 mg/kg, Observation time: 24 h, 5 male and 5 female

Control animals:

yes

Positive control(s):

Name: cyclophosphamide
Supplier: SIGMA CHMIE Gmbh
- Route of administration: intraperitoneal
- Doses / concentrations: 80 mg/kg
- Application volume: 0.1 ml/10 g body weight
- Solvent: water for injection

The preparations of the positive control substance were freshly prepared before administration.

Tissues and cell types examined:

The cells suspension was obtained from the extraction of bone marrow from the medullary canal of femurs.

Details of tissue and slide preparation:

The cells suspension was treated and the specimens colored following Pappenheimer's method.
2000 polychromatic erythrocytes per animal were examined microscopically in order to determine the presence and the number o micronuclei.

Evaluation criteria:

The classification of micronuclei was performed using the following criteria:
- Micronuclei are round, rarely oval or crescent shaped.
- They have a sharp contour.
- They are uniformly colored and are approximately from 1/20 to 1/5 of the diameter of the erythrocytes.
- It is usually only a micronucleus present.
The ratio of polychromatic to normochromatic erythrocytes was determined individually for each animal by counting a total of 1000 erythrocytes.

Statistics:

The statistics of the test were riported in the study report.
The calculations were performed using the program MUTAI (SOP M/EDV/036) performed on a 386 AT DIGICOM. The program is written in PowerBASIC and is extensively tested.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

No signs of systemic intoxication were observed in female mice for the sigle oral doses of 1600, 800 and 400 mg/kg.
The highest dose of 1600 mg/kg causes the death of 2 male mice within 24h. Conseguently the dose was lowered to 1200 mg/kg. The other doses of 800 and 400 mg/kg were tolerate without symptoms.

The results show no citotoxic effect of any chromosomal damages.

Conclusions:

Interpretation of results: negative
No cytotoxic effect of the test substance were observed.
No potential of chromosomal damages were observed.

Executive summary:

This end point was assessed following the OECD 474 and it was performed in GLP conditions. Micronuclei arise as a result of damage to the chromosomes or the spindle apparatus through a mutagenic active substance during the mitotic cell division in proliferating bone marrow cells. The results show no increased incidence of micronucleated polychromatic erythrocytes

The calculated frequency of the micronuleated polychromatic erythrocytes of the groups treated with the substance was between 0,17 and 0,42 %.The observed frequency corresponds to the data reported in the literature which is related to the spontaneous rate of occurrence of micronuclei in polychromatic erythrocytes.

Hence a potential of chromosomal damages or damage to the mitotic apparatus could be excluded for the tested substance.

Reference 2

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

other: experimental results on similar substance

Adequacy of study:

key study

Study period:

15. Jul 1996 - 16. Oct 1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

according to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

GLP compliance:

yes

Type of assay:

unscheduled DNA synthesis

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Karl Thomae GmbH, Biberach an der Riss, Germany.
- Weight at study initiation: 254g (mean)
- Housing: Makrolon cages, type III
- Diet: Standardized pelleted feed (Kliba-Haltungsdiät/Provimi Kliba SA, Kaiseraugst, Switzerland) was available ad libitum.
- Water: drinking water from bottles was available ad libitum.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 °C
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12 hours light from 6.00 - 18.00 hours and 12 hours darkness from 18.00 - 6.00 hours). During the time between treatment and perfusion the day/night rhythm was not followed.

Route of administration:

oral: unspecified

Vehicle:

- Vehicle/solvent used: CMC (carboxymethyl cellulose).
- Justification for choice of solvent/vehicle: Due to the Iimited solubility of the test substance in water, a 0.5% CMC formulation was selected as the vehicle.
- Concentration of test material in vehicle: Test group 3 and 4: 5g/100ml; Test group 5 and 6: 10g/100ml

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: To achieve homogeneity of the test substance in the vehicle, the test substance formulation was stirred constantly during removal and administration.

Duration of treatment / exposure:

12 and 18h

Frequency of treatment:

The treatment consisted of a single oral administration.

Remarks:

Doses / Concentrations:
500 mg/kg
Basis:
nominal conc.
dose group 3 and 4

Remarks:

Doses / Concentrations:
1000 mg/kg
Basis:
nominal conc.
dose group 5 and 6

No. of animals per sex per dose:

3

Control animals:

yes, historical

Positive control(s):

2-acetylaminofluorene;
- Justification for choice of positive control(s): 2-acetylaminofluorene is a well-established UDS-inducing agent.
- Route of administration: oral
- Doses / concentrations: 50 mg/kg

Tissues and cell types examined:

Isolated primary rat hepatocytes.

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION: Culture conditions (seeding and attachment period):
The isolated hepatocytes were seeded on coverslips on 1.9 cm² well containing 2 ml of attachment medium (WMEC).
- about 400,000 viable cells were seeded per well.
- 6 wells/per animal were used for the UDS assay.
After an attachment period of about 2 hours with 5% CO2 at 37°C and > 90% humidity, the medium (WMEC) was replaced by fresh medium (WMF 1) to remove non-adherent celIs.

Labeling
The medium (WMEI) was replaced by 2 ml labeling medium, and the cells were incubated with 5% CO2 at 37°C and > 90% humidity for 4 hours. After the labeling period, cells were washed with HBSS or WMEI (about 37°C); then fresh medium containing 0.25 mM unlabeled thymidine was added and the cells were incubated for another 14 hours. The cells on the coverslips were then fixed with ethanol/acetic acid (3 :1, v/v) for at least 30 minutes, rinsed 2 - 4 times with aqua dest. and air-dried. The dried coverslips were mounted ceIl side up on glass slides using Corbit-Balsam and dried overnight.

Autoradiography:
The slides were coated with KODAK NTB-2 photographic emulsion (about 37°C) for about 5 - 10 seconds. After drying at room temperature in the dark (Iightproof boxes) for about 16 hours, the coated slides were started in the dark with a desiccant (in the presence of a drying agent) at -20°C for 2 -10 days. Thereafter, the slide boxes were left at room temperature for at least 3 hours. The photographic emulsion was then developed with KODAK D-19 (about 15°C), fixed in KODAK Acidofix for about 5 minutes, washed in water for about 5 -10 minutes and stained with methyl green-pyronine Y. After rinsing with water and ethanol and air-drying, the slides were covered with a second coverslip using Corbit-Balsam.


METHOD OF ANALYSIS:
Quantification of UDS/Microscopic evaluation:
The quantification of UDS was performed microscopically generally using 3 slides per test group. 25 - 50 cells in good morphological conditions were randomly selected per slide and examined to achieve a total number of 100 cells/animal. For each celI, the following counts were performed with an automatic image analyzer (ARTEK):
- the nuclear grain (NG) count (= number of silver grains overlying the nucleus)
- the cytoplasmic grain (GG) count (= number of grains in two or three nucleusequivalent areas adjacent to the nucleus).
The following parameters were calculated:
- the net nuclear grain (NNG) count of each cell (= nuclear grain count minus
cytoplasmic grain count; NG - GG)
- the mean nuclear grain (NG) count
- the mean cytoplasmic grain (GG) count
- the mean net nuclear grain (NNG) count
- the percentage of cells in repair (cells showing net nuclear grain counts of > 0)
- the percentage of cells in repair (cells showing net nuclear grain counts of > 5)
Slides were coded before microscopic evaluation.

Evaluation criteria:

The test substance was considered positive if a dose-related increase was demonstrated in both of the following:
- The mean number of net nuclear grain (NNG) counts, which must exceed zero at one of the test points.
- The percentage of cells in repair (NNG > 5) when > 20.
A dose-related increase in % cells in repair > 5 outside the values of both the concurrent negative control and the historical control data base (> 5 < 20) and a dose-related increase in the mean number of NNG counts near to but without exceeding zero is considered to be an indication for a marginal response which needs to be confirmed/clarified in a further experiment. A test article producing both NNG counts and % cells in repair in the range of the negative control data is considered to be negative in the in vitro UDS assay.

Statistics:

Due to very clear negative findings, a statistical evaluation was not carried out.

Sex:

male

Genotoxicity:

negative

Remarks:

(500 mg/kg and 1000 mg/kg)

Toxicity:

no effects

Remarks:

no cytotoxicity noted (trypan blue exclusion technique)

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

After the single oral administration of the test substance preparation, all animals showed symptoms in form of:
-piloerection
-orange discoloration of for- and hindlimbs, tail and ears
-yellow to brownish discoloration of the lymphatic organs, the adiposal tissue and the liver
-anogenital region smeared with feces and test substance
The urine of the animals was discolored orange/darkbrown.
Furthermore, 5 out of 11 animals in the 1,000 mg/kg group died.

DNA repair activity

Perfusion 12 hours after treatment (mean of 3 animals ± standard deviation)

Test Group	Vehicle control	500 mg/kg	1000 mg/kg	positive control
NG counts	6.53± 1.84	7.06 ±0.70	8.19±1.89	18.11 ±4.44
CG counts	10.17±2.04	10.73±0.77	10.41 ±1.26	11.23 ±1.60
NNG counts	-3.64 ±0.47	-3.67 ±0.62	-2.21 ±2.45	6.88 ±2.88
% cells in repair NGG > 0	9.33 ± 4.51	7.00 ± 4.36	20.00 ± 18.19	78.67 ±7.57
% cells in repair NGG > 5	0.00 ± 0.00	0.00 ± 0.00	5.67 ± 8.96	54.00 ±11.53

NG = nuclear grains

CG = cytoplasmic grains

NNG = net nuclear grains

Perfusion 18 hours after treatment (mean of 3 animals ± standard deviation)

Test Group	Vehicle control	500 mg/kg	1000 mg/kg	positive control
NG counts	6.35 ± 0.36	6.54 ± 0.54	7.48±0.88	20.43 ± 1.63
CG counts	11.08 ±0.30	9.90 ± 0.85	10.94±1.16	11.88 ±0.56
NNG counts	-4.73 ±0.65	-3.36 ± 0.59	-3.45 ±0.53	8.56 ±1.34
% cells in repair NGG > 0	7.67 ± 4.04	15.00 ± 4.00	14.67 ±4.73	91.67 ±4.04
% cells in repair NGG > 5	0.00 ± 0.00	0.33 ±0.58	0.67 ±0.58	65.67 ±9.50

NG = nuclear grains

CG = cytoplasmic grains

NNG = net nuclear grains

mean per group (mean of 3 animals)

Conclusions:

The test substance did not cause a relevant increase in unscheduled DNA synthesis, as measured by an increase in net nuclear grain counts, and it was concluded that the test substance was negative in this in vivo assay using primary rat hepatocytes.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Both in vitro and in vivo mutagenicity tests are available for Direct Black 19.

Bacterial reverse mutation assay, Ames test was positive for some strains.

The in vitro gene mutation test, mammalian cell gene mutation assay, did not show any mutagenic effect.

The results of the in vivo micronucleous assay, chromosome aberration, are negative.

In addiction also a test for unscheduled DNA synthesis, damage and/or repair, for a similar substance showed negative results.

Based on these tests, the Direct Black 19 could be considered as not mutagenic.

Justification for classification or non-classification

GERM CELL MUTAGENICITY

This hazard class is primarily concerned with substances that may cause mutations in the germ cells of humans that can be transmitted to the progeny. However, the results from mutagenicity or genotoxicity tests in vitro and in mammalian somatic and germ cells in vivo are also considered in classifying substances and mixtures within this hazard class.

Category 1: Substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans. Substances known to induce heritable mutations in the germ cells of humans.

Categoty 2: Substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans.

The classification in Category 2 is based on:

— positive evidence obtained from experiments in mammals and/or in some cases from in vitro experiments, obtained from:

— somatic cell mutagenicity tests in vivo, in mammals; or

— other in vivo somatic cell genotoxicity tests which are supported by positive results from in vitro mutagenicity assays.

Note: Substances which are positive in in vitro mammalian mutagenicity assays, and which also show chemical structure activity relationship to known germ cell mutagens, shall be considered for classification as Category 2 mutagens.

Classification for heritable effects in human germ cells is made on the basis of well conducted, sufficiently validated tests as:

- In vivo somatic cell mutagenicicty tests such as these indicated in paragraph 3.5.2.3.5:

— mammalian bone marrow chromosome aberration test;

— mouse spot test;

— mammalian erythrocyte micronucleus test.

- In vitro mutagenicity tests such as these indicated in 3.5.2.3.8:

— in vitro mammalian chromosome aberration test;

— in vitro mammalian cell gene mutation test;

— bacterial reverse mutation tests.

Therefore, based on the available in vitro and in vivo studies, it is possible to conclude that the test substance is Not Classified for Mutagenic Toxicity.