Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Not mutagenic

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
other: experimentakresult on similar substance
Adequacy of study:
key study
Study period:
1993
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
Pre-incubation test. Screening program: strain TA98 placed in standard plates. Evaluation without metabolic activation.
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 98
Metabolic activation:
without
Metabolic activation system:
No
Test concentrations with justification for top dose:
Range 20-5000 µg/plates: 20, 100, 500, 2500, 5000 µg/plate
Untreated negative controls:
yes
Remarks:
DMSO
Positive controls:
yes
Remarks:
NPD
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: 2500-5000 µg/plate

Revertant/plate Quotient
 Dose µg/plate - S9  M DS

-S9

 Negative control

29

29

18

25  6 1.0
20

19

13

24

19 6 0.7
100

26

26

26

26 0 1.0
500

31

29

28

29 1.2
2500

31

30

24

28 4 1.1
5000

32

31

33

32 1 1.3 
Positive control

608

572

666

615  47  24.3 
Conclusions:
Negative
Executive summary:

Non mutagen; no increase of revertants has been observed.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
other: experimental resulta on similar substance
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
Remarks on result:
other: strain/cell type: cell line V79, clone 65/3
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Not mutagenic

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
other: experimental resulta on similar substance
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 (migrated information): 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.



Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

Not mutagenic based on available data both in vitro and in vivo.