Registration Dossier

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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial Gene Mutation


PR012


The test item did not exert mutagenic activity in the reverse bacterial mutation assay (plate incorporation assay and pre-incubation assay according to Prival) with and without metabolic activation.


Chromosome Aberration


PR022


In a guideline study (OECD 473) the test item, suspended in ethanol, was assessed for its potential to induce structural chromosome aberrations in Chinese hamster lung fibroblasts (CHL/IU) with and without metabolic activation.


It was concluded that the test substance is not clastogenic in Chinese hamster lung fibroblast cells (CHL/IU) under the experimental conditions described in the report.


Mammalian Gene Mutation


PR112


The test item was not genotoxic in the mammalian cell gene (HPRT) mutation test in V79 Chinese Hamster cells when tested with and without rat liver S9 metabolic activation at concentrations up to 400 µg/ml.


In vitro micronucleus


PR112


In an in-vitro micronuclus assay in Chinese hamster V79 cells, the test item at concentrations up to 31.3 µg/ml did not induce micronuclei in the absence and presence of metabolic activation. Therefore, Pigment Red 112 can be considered as non-mutagenic in this in vitro test system, when tested up to precipitating concentrations.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD 473) and according to GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
other: Chinese hamster lung cells (CHL/IU)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: no data
Metabolic activation:
with and without
Metabolic activation system:
liver S9 mix from phenobarbital and 5,6-benzoflavone induced rats
Test concentrations with justification for top dose:
cell growth inhibition test: 0, 8.4, 16.8, 33.6, 67.2, 134.4, 268.8, 537.5, 1075, 2150, 4300 µg/mL
chromosomal abberation test: 0, 37.5, 75, 150, 300, 600 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: test substance is insolube in water, DMSO and acetone
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
for continuous treatment and short treatment in absence of metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: dimethylnitrosamine
Remarks:
for short treatment method in the presence of metabolic activation
Details on test system and experimental conditions:
DURATION
- Exposure duration:
continuous treatment: 24 or 48 h
short treatment: 6 h
- Expression time (cells in growth medium):
short treatment: 18 h
- Fixation time (start of exposure up to fixation or harvest of cells):
continuous treatment: 24 or 48 h
short treatment: 24h

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (2 h before completion of incubation)
STAIN (for cytogenetic assays): Giemsa solution

NUMBER OF REPLICATIONS: Doubling time: 16.2 h

NUMBER OF CELLS EVALUATED: 200 cells per concentration

DETERMINATION OF CYTOTOXICITY
- Method: cell survival ratio
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
Evaluation criteria:
The final judgement on the outcome of the test was made as follows: when the incidence of cells with numerical or structural aberrations induced by the test article was lower than 5, the result was assessed as negative; from 5-10% (exclusive of 10%), equivocal; and 10% or higher with a concentration-dependent rise, positive. The incidence of cells having structural abberations with gaps and of cells without gaps were calculated separately, and cells without gaps were assessed for chromosomal aberrarions.
Statistics:
No significance tests were performed, since the incidence of cells with chromosomal aberrations was assessed according to the evaluation criteria described above.
Species / strain:
other: Chinese hamster lung cells (CHL/IU)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
precipitation occurred at concentrations of 268.8 µg/mL and higher
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: precipitation occurred at concentrations of 268.8 µg/mL and higher

ADDITIONAL INFORMATION ON CYTOTOXICITY:
in the cell growth inhibition test no cytotoxicity was noted.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

The test item is not clastogenic in Chinese hamster lung fibroblast (CHL/IU) cells under the experimental conditions described in the study.
Executive summary:

In a guideline study (OECD 473) the test item, suspended in ethanol, was assessed for its potential to induce structural chromosome aberrations in Chinese hamster lung fibroblasts (CHL/IU) with and without metabolic activation.

Prior to the chromosomal aberration test, a cell growth inhibition test was performed to determine test concentrations for the chromosomal aberration test. No cytotoxicity was noted at 4300 µg/mL, the highest concentration tested. Precipitations were noted at concentrations of 268.8 µg/mL and higher.

In the chromosomal aberration test, concentrations of 0, 37.5, 75, 150, 300 and 600 µg/mL were tested. A continuous exposure (24 or 48 h) and a short time exposure (6 h with 18 h expression time) in the presence and absence of S9-mix were performed.

At least 200 metaphases per culture were evaluated for structural chromosome aberrations.

Appropriate mutagens were used as positive controls. They produced markedly positive results.

It was concluded that the test substance is not clastogenic in Chinese hamster lung fibroblast cells (CHL/IU) under the experimental conditions described in the report.

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: According to Draft Proposal for a new OECD Guideline 487 and GLP
Justification for type of information:
See rationale and justification for the analogue read-across approach for the registration of the nanoform of Pigment Red 012 (chapter 13).
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Principles of method if other than guideline:
OECD Guideline Draft Proposal for a new Guideline No. 487, Version 3
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
Micronucleus
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
Thawed stock cultures were propagated at 37 °C in 80 cm2 plastic flasks (Greiner,
72632 Frickenhausen, Germany). About 5 x 105 cells per flask were seeded in 15 mL of MEM (minimal essential medium; Invitrogen GIBCO, 76131 Karlsruhe, Germany) supplemented with 10 % fetal calf serum (FCS; Invitrogen, 76131 Karrlsruhe, Germany). Additionally, the medium was supplemented with 1 % 100x Penicillin/ Streptomycin solution (10.000 Units/mL Penicillin, 10 mg/mL Streptomycin; PAA Laboratories GmbH, 35091 Cölbe, Germany) and 1 % Amphotericin B (250 µg/mL, PAA Laboratories GmbH, 35091 Cölbe, Germany). The cells were subcultured twice weekly. The cell cultures were incubated at 37 °C in a humidified atmosphere with 4.5 % carbon dioxide (95.5% air).
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9
Test concentrations with justification for top dose:
Exp. I: with and without S9 mix: 2.0, 3.9, 7.8, 15.6, 31.3, 62.5, 125.0 and 250.0 µg/mL
Exp. II: with S9 mix: 0.3, 0.5, 1.0, 2.0, 3.9, 7.8, 15.6 and 31.3 µg/mL
without S9 mix: 0.5, 1.0, 2.0, 3.9, 7.8, 15.6, 31.3 and 62.5 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent: regard to the ability to formulate a manageable suspension of the test item in DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Migrated to IUCLID6: in the absence of S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Migrated to IUCLID6: in the presence of S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: griseofulvin - in the absence of S9 mix
Details on test system and experimental conditions:
Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without metabolic activation. In Experiment II the exposure period was 24 hours without S9 mix and 4 hours with metabolic activation. The cells were prepared 24 hours after start of treatment with the test item.

METHOD OF APPLICATION: in minimal essential medium

DURATION
- Exposure duration: 4 and 24 hours
- Expression time (cells in growth medium): 24 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours

SPINDLE INHIBITOR (cytogenetic assays):
STAIN (for cytogenetic assays): May Gruenwald and Giemsa

NUMBER OF REPLICATIONS: 1.5 - 2

NUMBER OF CELLS EVALUATED: 2000

DETERMINATION OF CYTOTOXICITY
- Method: Proliferation Index

OTHER: none




Evaluation criteria:
Evaluation of the cultures was performed manually using NIKON microscopes with 40 x oil immersion objectives. The micronuclei were counted in cells showing a clearly visible cytoplasm area. The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle. The micronuclei were stained in the same way as the main nucleus. The area of the micronucleus did not extend the third part of the area of the main nucleus. 2000 cells from clones with 2 - 8 cells were scored per test group. The frequency of micronucleated cells was reported as % micronucleated cells.
Statistics:

Statistical significance can be confirmed by means of the Chi square test.
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:
The test item, suspended in DMSO, was assessed for its potential to induce micronuclei in Chinese hamster V79 cells in vitro in the absence and presence of metabolic activation by S9 mix.
Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without metabolic activation. In Experiment II the exposure period was 24 hours without metabolic activation and 4 hours with metabolic activation. The cells were prepared 24 hours after start of treatment with the test item.
In each experimental group two parallel cultures were set up and 1000 cells per culture were scored for micronuclei.
No influence of the test item on pH value or osmolarity was observed (Exp. I: solvent control 314 mOsm, pH 7.8 versus 354 mOsm and pH 7.8 at 250.0 µg/mL; Exp. II: solvent control 365 mOsm, pH 7.7 versus 369 mOsm and pH 7.7 at 62.5 µg/mL).
In Experiment I test item precipitation in culture medium at the end of treatment was observed at 31.3 µg/mL and above in the absence and presence of metabolic activation. In Experiment II precipitation occurred at 15.6 µg/mL and above in the absence of metabolic activation and at 31.3 µg/mL and above in the presence of metabolic activation.
On the evaluated slides neither in the absence nor in the presence of metabolic activation cytotoxicity measured as reduced proliferation index was observed up to the highest evaluable concentration.
In the absence and presence of metabolic activation no statistically significant or biologically relevant increase in the percentage of micronucleated cells was observed up to the highest evaluable concentrations. The rates of micronucleated cells after treatment with the test item (0.20 - 1.50 %) were close to the range of the solvent control values (0.35 – 1.25 %) and within the range of the laboratory´s historical control data.
Mitomycin C (0.1 µg/mL), Griseofulvin (9.0 µg/mL) and CPA (10.0 and 15.0 µg/mL) were used as positive controls and showed a distinct increase in the percentage of micronucleated cells.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Summary of results of the micronucleus test with test item

Exp.

Preparation

Test item

Proliferation

Micronucleated

interval

concentration

Index

Cells*

in µg/mL

in %

Exposure period 4 hrs without S9 mix

I

24 hrs

Solvent control1

2.92

 0.35

Positive control2

2.60

 6.90S

7.8

2.95

 0.35

15.6

3.02

 0.45

31.3P

2.95

 0.65

Exposure period 24 hrs without S9 mix

II

24 hrs

Solvent control1

3.04

 1.25

Positive control2

2.58

11.05S

Positive control3

2.55

24.20S

3.9

3.08

 0.70

7.8

2.99

 1.15

15.6P

2.97

 1.50

Exposure period 4 hrs with S9 mix

I

24 hrs

Solvent control1

2.53

 0.75

Positive control4

1.86

 4.95S

2.0

2.61

 0.55

3.9

2.69

 0.70

7.8

2.67

 0.20

II

24 hrs

Solvent control1

2.09

 0.95

Positive control5

1.66

 7.25S

2.0

2.04

 0.50

3.9

2.11

 0.40

7.8

2.00

 0.25

*     The number of micronucleated cells was determined of each test group in a sample of 2000 cells

P      Precipitation occurred at the end of treatment

S     Number of micronucleated cells statistically significantly higher than corresponding control values

1           DMSO       0.5% (v/v)

2           Mitomycin C        0.1 µg/mL

3           Griseofulvin 9.0 µg/mL

4           CPA   10.0 µg/mL

5           CPA   15.0 µg/mL

Conclusions:
In an in-vitro micronuclus assay in Chinese hamster V79 cells, the test item at concentrations up to 31.3 µg/ml did not induce micronuclei in the absence and presence of metabolic activation. Therefore, Pigment Red 112 can be considered as non-mutagenic in this in vitro test system, when tested up to precipitating concentrations.
Executive summary:

The test item, suspended in DMSO, was assessed for its potential to induce micronuclei in Chinese hamsterV79cells in vitro in the absence and presence of metabolic activation by S9 mix. The following study design was performed:

Experiment I

Experiment II

Without S9 mix

With S9 mix

Without S9 mix

With S9 mix

Exposure period

 4 hours

 4 hours

24 hours

 4 hours

Recovery

20 hours

20 hours

 0 hours

20 hours

Preparation interval

24 hours

24 hours

24 hours

24 hours

In each experimental group two parallel cultures were analysed and 1000 cells per culture were scored for micronuclei.

The highest applied concentration (250 µg/mL) was chosen with respect to the ability to formulate a homogeneous suspension of the test item in DMSO. The following test item concentrations were applied:

Exp. I:   with and without S9 mix: 2.0, 3.9, 7.8, 15.6, 31.3, 62.5, 125.0 and 250.0 µg/mL

Exp. II:             without S9 mix:             0.5, 1.0, 2.0, 3.9, 7.8, 15.6, 31.3 and 62.5 µg/mL
             with S9 mix: 0.3, 0.5, 1.0, 2.0, 3.9, 7.8, 15.6 and 31.3 µg/mL.

On the evaluable slides neither in the absence nor in the presence of metabolic activation cytotoxicity measured as reduced proliferation index was observed. Due to strong test item precipitation in culture or on the slides higher concentrations could not be evaluated for cytogenetic damage.

In the presence as well as in the absence of metabolic activation, no biologically relevant increase in the percentage of micronucleated cells was observed after treatment with the test item with respect to the evaluation criteria mentioned in the current guideline forin vitrogenotoxicity studies.

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in the percentage of micronucleated cells.

In conclusion, it can be stated that under the experimental conditions reported, the test item Pigment Red 112 did not induce micronuclei in V79 cells (Chinese hamster cell line) in vitro in the absence and presence of metabolic activation.

Therefore, Pigment Red 112 is considered to be non-mutagenic in thisin vitrotest system, when tested up to precipitating concentrations.

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: According to Draft Proposal for a new OECD Guideline 487 and GLP
Qualifier:
according to guideline
Principles of method if other than guideline:
OECD Guideline Draft Proposal for a new Guideline No. 487, Version 3
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
Micronucleus
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
Thawed stock cultures were propagated at 37 °C in 80 cm2 plastic flasks (Greiner,
72632 Frickenhausen, Germany). About 5 x 105 cells per flask were seeded in 15 mL of MEM (minimal essential medium; Invitrogen GIBCO, 76131 Karlsruhe, Germany) supplemented with 10 % fetal calf serum (FCS; Invitrogen, 76131 Karrlsruhe, Germany). Additionally, the medium was supplemented with 1 % 100x Penicillin/ Streptomycin solution (10.000 Units/mL Penicillin, 10 mg/mL Streptomycin; PAA Laboratories GmbH, 35091 Cölbe, Germany) and 1 % Amphotericin B (250 µg/mL, PAA Laboratories GmbH, 35091 Cölbe, Germany). The cells were subcultured twice weekly. The cell cultures were incubated at 37 °C in a humidified atmosphere with 4.5 % carbon dioxide (95.5% air).
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9
Test concentrations with justification for top dose:
Exp. I: with and without S9 mix: 2.0, 3.9, 7.8, 15.6, 31.3, 62.5, 125.0 and 250.0 µg/mL
Exp. II: with S9 mix: 0.3, 0.5, 1.0, 2.0, 3.9, 7.8, 15.6 and 31.3 µg/mL
without S9 mix: 0.5, 1.0, 2.0, 3.9, 7.8, 15.6, 31.3 and 62.5 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent: regard to the ability to formulate a manageable suspension of the test item in DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Migrated to IUCLID6: in the absence of S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Migrated to IUCLID6: in the presence of S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: griseofulvin - in the absence of S9 mix
Details on test system and experimental conditions:
Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without metabolic activation. In Experiment II the exposure period was 24 hours without S9 mix and 4 hours with metabolic activation. The cells were prepared 24 hours after start of treatment with the test item.

METHOD OF APPLICATION: in minimal essential medium

DURATION
- Exposure duration: 4 and 24 hours
- Expression time (cells in growth medium): 24 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours

SPINDLE INHIBITOR (cytogenetic assays):
STAIN (for cytogenetic assays): May Gruenwald and Giemsa

NUMBER OF REPLICATIONS: 1.5 - 2

NUMBER OF CELLS EVALUATED: 2000

DETERMINATION OF CYTOTOXICITY
- Method: Proliferation Index

OTHER: none




Evaluation criteria:
Evaluation of the cultures was performed manually using NIKON microscopes with 40 x oil immersion objectives. The micronuclei were counted in cells showing a clearly visible cytoplasm area. The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle. The micronuclei were stained in the same way as the main nucleus. The area of the micronucleus did not extend the third part of the area of the main nucleus. 2000 cells from clones with 2 - 8 cells were scored per test group. The frequency of micronucleated cells was reported as % micronucleated cells.
Statistics:

Statistical significance can be confirmed by means of the Chi square test.
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:
The test item, suspended in DMSO, was assessed for its potential to induce micronuclei in Chinese hamster V79 cells in vitro in the absence and presence of metabolic activation by S9 mix.
Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without metabolic activation. In Experiment II the exposure period was 24 hours without metabolic activation and 4 hours with metabolic activation. The cells were prepared 24 hours after start of treatment with the test item.
In each experimental group two parallel cultures were set up and 1000 cells per culture were scored for micronuclei.
No influence of the test item on pH value or osmolarity was observed (Exp. I: solvent control 314 mOsm, pH 7.8 versus 354 mOsm and pH 7.8 at 250.0 µg/mL; Exp. II: solvent control 365 mOsm, pH 7.7 versus 369 mOsm and pH 7.7 at 62.5 µg/mL).
In Experiment I test item precipitation in culture medium at the end of treatment was observed at 31.3 µg/mL and above in the absence and presence of metabolic activation. In Experiment II precipitation occurred at 15.6 µg/mL and above in the absence of metabolic activation and at 31.3 µg/mL and above in the presence of metabolic activation.
On the evaluated slides neither in the absence nor in the presence of metabolic activation cytotoxicity measured as reduced proliferation index was observed up to the highest evaluable concentration.
In the absence and presence of metabolic activation no statistically significant or biologically relevant increase in the percentage of micronucleated cells was observed up to the highest evaluable concentrations. The rates of micronucleated cells after treatment with the test item (0.20 - 1.50 %) were close to the range of the solvent control values (0.35 – 1.25 %) and within the range of the laboratory´s historical control data.
Mitomycin C (0.1 µg/mL), Griseofulvin (9.0 µg/mL) and CPA (10.0 and 15.0 µg/mL) were used as positive controls and showed a distinct increase in the percentage of micronucleated cells.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Summary of results of the micronucleus test with test item

Exp.

Preparation

Test item

Proliferation

Micronucleated

interval

concentration

Index

Cells*

in µg/mL

in %

Exposure period 4 hrs without S9 mix

I

24 hrs

Solvent control1

2.92

 0.35

Positive control2

2.60

 6.90S

7.8

2.95

 0.35

15.6

3.02

 0.45

31.3P

2.95

 0.65

Exposure period 24 hrs without S9 mix

II

24 hrs

Solvent control1

3.04

 1.25

Positive control2

2.58

11.05S

Positive control3

2.55

24.20S

3.9

3.08

 0.70

7.8

2.99

 1.15

15.6P

2.97

 1.50

Exposure period 4 hrs with S9 mix

I

24 hrs

Solvent control1

2.53

 0.75

Positive control4

1.86

 4.95S

2.0

2.61

 0.55

3.9

2.69

 0.70

7.8

2.67

 0.20

II

24 hrs

Solvent control1

2.09

 0.95

Positive control5

1.66

 7.25S

2.0

2.04

 0.50

3.9

2.11

 0.40

7.8

2.00

 0.25

*     The number of micronucleated cells was determined of each test group in a sample of 2000 cells

P      Precipitation occurred at the end of treatment

S     Number of micronucleated cells statistically significantly higher than corresponding control values

1           DMSO       0.5% (v/v)

2           Mitomycin C        0.1 µg/mL

3           Griseofulvin 9.0 µg/mL

4           CPA   10.0 µg/mL

5           CPA   15.0 µg/mL

Conclusions:
In an in-vitro micronuclus assay in Chinese hamster V79 cells, the test item at concentrations up to 31.3 µg/ml did not induce micronuclei in the absence and presence of metabolic activation. Therefore, Pigment Red 112 can be considered as non-mutagenic in this in vitro test system, when tested up to precipitating concentrations.
Executive summary:

The test item, suspended in DMSO, was assessed for its potential to induce micronuclei in Chinese hamsterV79cells in vitro in the absence and presence of metabolic activation by S9 mix. The following study design was performed:

Experiment I

Experiment II

Without S9 mix

With S9 mix

Without S9 mix

With S9 mix

Exposure period

 4 hours

 4 hours

24 hours

 4 hours

Recovery

20 hours

20 hours

 0 hours

20 hours

Preparation interval

24 hours

24 hours

24 hours

24 hours

In each experimental group two parallel cultures were analysed and 1000 cells per culture were scored for micronuclei.

The highest applied concentration (250 µg/mL) was chosen with respect to the ability to formulate a homogeneous suspension of the test item in DMSO. The following test item concentrations were applied:

Exp. I:   with and without S9 mix: 2.0, 3.9, 7.8, 15.6, 31.3, 62.5, 125.0 and 250.0 µg/mL

Exp. II:             without S9 mix:             0.5, 1.0, 2.0, 3.9, 7.8, 15.6, 31.3 and 62.5 µg/mL
             with S9 mix: 0.3, 0.5, 1.0, 2.0, 3.9, 7.8, 15.6 and 31.3 µg/mL.

On the evaluable slides neither in the absence nor in the presence of metabolic activation cytotoxicity measured as reduced proliferation index was observed. Due to strong test item precipitation in culture or on the slides higher concentrations could not be evaluated for cytogenetic damage.

In the presence as well as in the absence of metabolic activation, no biologically relevant increase in the percentage of micronucleated cells was observed after treatment with the test item with respect to the evaluation criteria mentioned in the current guideline forin vitrogenotoxicity studies.

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in the percentage of micronucleated cells.

In conclusion, it can be stated that under the experimental conditions reported, the test item Pigment Red 112 did not induce micronuclei in V79 cells (Chinese hamster cell line) in vitro in the absence and presence of metabolic activation.

Therefore, Pigment Red 112 is considered to be non-mutagenic in thisin vitrotest system, when tested up to precipitating concentrations.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
29 January - 14 March 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to OECD test guideline and GLP
Justification for type of information:
See rationale and justification for the analogue read-across approach for the registration of the nanoform of Pigment Red 012 (chapter 13).
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
hypoxanthine-guanine phosphoribosyl transferase
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: minimum essential medium containing 10% fetal calf serum
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (phenobarbital/beta-naphthoflavone-induced rat liver protein with cofactors)
Test concentrations with justification for top dose:
3.1, 6.3, 12.5, 25.0, 50.0 and 400 µg/ml with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Migrated to IUCLID6: w/o metabolic activation; 7,12-Dimethylbenz(a)anthracene with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium (4h-incubation without serum, 24 h incubations with serum)


DURATION
- Preincubation period: none
- Exposure duration: 4 and 24 h
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 8 days
- Fixation time (start of exposure up to fixation or harvest of cells):


SELECTION AGENT (mutation assays): 6-thioguanine
STAIN (for cytogenetic assays): 10% methylene blue in 0.01% KOH solution


NUMBER OF REPLICATIONS: 5/experiment, 2 experiments


NUMBER OF CELLS EVALUATED: colonies >50 cells


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:
test item classified as positive for mutagenicity if induces either concentration-related increase of mutant frequency or reproducible and positive response at one of the test points (at least three times above the spontaneous mutation frequency)
Statistics:
linear regerssion on dose-dependeny of mutant frequencies using SYSTAT statistics software
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
precipitation was observed at 12.5 µg/ml and higher (Exp. I without S9-mix), at 25.0 µg/ml and higher (Exp. I with S9-mix and Exp. II)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: negligible decrease (0.1 pH-units) at highest dose
- Effects of osmolality: negligible increase (+3 mOsm) at highest dose
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Results of V79/HPRT assay with test item

Concentration [microgram/mL]

mutant colonies per 10^-6 cells**

mutant colonies per 10^6 cells**

Cytotoxicity
(yes/no)

Remarks

 

— MA

+ MA

 

 

0*

 7.6, 6.2

 5.2, 21.2

 no

 Exp. I

3.1

13.6, 8.5

 11.7, 13.4

 no

 

6.3

 7.8, 14.7

 8.6, 26.8

 no

 

12.5

12.3, 8.6

 13.4, 19.5

 no

 

25.0

 12.3, 10.9

 7.4, 22.3

 no

400

 15.9, 10.6

 13.1, 12.5

no 

 

Positive control***

151.7, 87.5

 1239.9, 1528.8

 -MA: no;

+MA: yes

 

0*

13.9, 11.3

 -

 no

 Exp. II

3.1

11.4, 6.1

 -

 no

 

6.3

12.2, 7.7

 -

 no

 

12.5

17.1, 12.9

 -

 no

 

25.0

11.0, 9.6

-

 no

 

400

5.5, 7.7

 -

 no

 

Positive control***

186.6,. 136.9

 -

 yes

 

*solvent control with DMSO

** mean of 5 plates each in culture I and culture II

***without MA: EMS, with MA: DMBA

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

The test item was not genotoxic in the mammalian cell gene (HPRT) mutation test in V79 Chinese Hamster cells when tested with and without rat liver S9 metabolic activation at concentrations up to 400 µg/ml.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD 473) and according to GLP.
Justification for type of information:
See rationale and justification for the analogue read-across approach for the registration of the nanoform of Pigment Red 012 (chapter 13).
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
other: Chinese hamster lung cells (CHL/IU)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: no data
Metabolic activation:
with and without
Metabolic activation system:
liver S9 mix from phenobarbital and 5,6-benzoflavone induced rats
Test concentrations with justification for top dose:
cell growth inhibition test: 0, 8.4, 16.8, 33.6, 67.2, 134.4, 268.8, 537.5, 1075, 2150, 4300 µg/mL
chromosomal abberation test: 0, 37.5, 75, 150, 300, 600 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: test substance is insolube in water, DMSO and acetone
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
for continuous treatment and short treatment in absence of metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: dimethylnitrosamine
Remarks:
for short treatment method in the presence of metabolic activation
Details on test system and experimental conditions:
DURATION
- Exposure duration:
continuous treatment: 24 or 48 h
short treatment: 6 h
- Expression time (cells in growth medium):
short treatment: 18 h
- Fixation time (start of exposure up to fixation or harvest of cells):
continuous treatment: 24 or 48 h
short treatment: 24h

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (2 h before completion of incubation)
STAIN (for cytogenetic assays): Giemsa solution

NUMBER OF REPLICATIONS: Doubling time: 16.2 h

NUMBER OF CELLS EVALUATED: 200 cells per concentration

DETERMINATION OF CYTOTOXICITY
- Method: cell survival ratio
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
Evaluation criteria:
The final judgement on the outcome of the test was made as follows: when the incidence of cells with numerical or structural aberrations induced by the test article was lower than 5, the result was assessed as negative; from 5-10% (exclusive of 10%), equivocal; and 10% or higher with a concentration-dependent rise, positive. The incidence of cells having structural abberations with gaps and of cells without gaps were calculated separately, and cells without gaps were assessed for chromosomal aberrarions.
Statistics:
No significance tests were performed, since the incidence of cells with chromosomal aberrations was assessed according to the evaluation criteria described above.
Species / strain:
other: Chinese hamster lung cells (CHL/IU)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
precipitation occurred at concentrations of 268.8 µg/mL and higher
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: precipitation occurred at concentrations of 268.8 µg/mL and higher

ADDITIONAL INFORMATION ON CYTOTOXICITY:
in the cell growth inhibition test no cytotoxicity was noted.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

The test item is not clastogenic in Chinese hamster lung fibroblast (CHL/IU) cells under the experimental conditions described in the study.
Executive summary:

In a guideline study (OECD 473) the test item, suspended in ethanol, was assessed for its potential to induce structural chromosome aberrations in Chinese hamster lung fibroblasts (CHL/IU) with and without metabolic activation.

Prior to the chromosomal aberration test, a cell growth inhibition test was performed to determine test concentrations for the chromosomal aberration test. No cytotoxicity was noted at 4300 µg/mL, the highest concentration tested. Precipitations were noted at concentrations of 268.8 µg/mL and higher.

In the chromosomal aberration test, concentrations of 0, 37.5, 75, 150, 300 and 600 µg/mL were tested. A continuous exposure (24 or 48 h) and a short time exposure (6 h with 18 h expression time) in the presence and absence of S9-mix were performed.

At least 200 metaphases per culture were evaluated for structural chromosome aberrations.

Appropriate mutagens were used as positive controls. They produced markedly positive results.

It was concluded that the test substance is not clastogenic in Chinese hamster lung fibroblast cells (CHL/IU) under the experimental conditions described in the report.

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
Study period:
29 January - 14 March 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to OECD test guideline and GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
hypoxanthine-guanine phosphoribosyl transferase
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: minimum essential medium containing 10% fetal calf serum
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (phenobarbital/beta-naphthoflavone-induced rat liver protein with cofactors)
Test concentrations with justification for top dose:
3.1, 6.3, 12.5, 25.0, 50.0 and 400 µg/ml with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Migrated to IUCLID6: w/o metabolic activation; 7,12-Dimethylbenz(a)anthracene with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium (4h-incubation without serum, 24 h incubations with serum)


DURATION
- Preincubation period: none
- Exposure duration: 4 and 24 h
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 8 days
- Fixation time (start of exposure up to fixation or harvest of cells):


SELECTION AGENT (mutation assays): 6-thioguanine
STAIN (for cytogenetic assays): 10% methylene blue in 0.01% KOH solution


NUMBER OF REPLICATIONS: 5/experiment, 2 experiments


NUMBER OF CELLS EVALUATED: colonies >50 cells


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:
test item classified as positive for mutagenicity if induces either concentration-related increase of mutant frequency or reproducible and positive response at one of the test points (at least three times above the spontaneous mutation frequency)
Statistics:
linear regerssion on dose-dependeny of mutant frequencies using SYSTAT statistics software
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
precipitation was observed at 12.5 µg/ml and higher (Exp. I without S9-mix), at 25.0 µg/ml and higher (Exp. I with S9-mix and Exp. II)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: negligible decrease (0.1 pH-units) at highest dose
- Effects of osmolality: negligible increase (+3 mOsm) at highest dose
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Results of V79/HPRT assay with test item

Concentration [microgram/mL]

mutant colonies per 10^-6 cells**

mutant colonies per 10^6 cells**

Cytotoxicity
(yes/no)

Remarks

 

— MA

+ MA

 

 

0*

 7.6, 6.2

 5.2, 21.2

 no

 Exp. I

3.1

13.6, 8.5

 11.7, 13.4

 no

 

6.3

 7.8, 14.7

 8.6, 26.8

 no

 

12.5

12.3, 8.6

 13.4, 19.5

 no

 

25.0

 12.3, 10.9

 7.4, 22.3

 no

400

 15.9, 10.6

 13.1, 12.5

no 

 

Positive control***

151.7, 87.5

 1239.9, 1528.8

 -MA: no;

+MA: yes

 

0*

13.9, 11.3

 -

 no

 Exp. II

3.1

11.4, 6.1

 -

 no

 

6.3

12.2, 7.7

 -

 no

 

12.5

17.1, 12.9

 -

 no

 

25.0

11.0, 9.6

-

 no

 

400

5.5, 7.7

 -

 no

 

Positive control***

186.6,. 136.9

 -

 yes

 

*solvent control with DMSO

** mean of 5 plates each in culture I and culture II

***without MA: EMS, with MA: DMBA

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

The test item was not genotoxic in the mammalian cell gene (HPRT) mutation test in V79 Chinese Hamster cells when tested with and without rat liver S9 metabolic activation at concentrations up to 400 µg/ml.
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:
From 10 MAR 2005 to 18 MAR 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD TG471) with Prival modification for azo-dyes
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Commission Directive 2000/32/EC, L1362000, Annex 4D
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 or hamster liver S9
Test concentrations with justification for top dose:
Experiment I (Plate incorporation method): 0, 3, 10, 33, 100, 333, 1000, 2500, 5000 µg/plate
Experiment II (Pre-incubation method): 0, 33, 100, 333, 1000, 2500, 5000 µg/plate
Vehicle / solvent:
- Vehicle/ Solvent used: DMSO
- Justification for choice of solvent/ vehicle: solubility properties and its relative non-toxicity to the bacteria
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide (TA 1535 and TA 100), 4-Nitro-o-phenylene-diamine (TA 1537 and TA 98), methyl methane sulfonate (WP2 uvrA)
Remarks:
without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with metabolic activation (rat liver S9 mix)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (TA 1535, TA 100, TA 1537 and WP2 uvrA), congo red (TA98)
Remarks:
with metabolic activation (hamster liver S9 mix)
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Experiment I: plate incorporation method without and with induced rat liver S9 mix (induction with phenobarbital/beta-naphthoflavone)
Experiment II: pre-incubation method without and with non-induced hamster liver S9 mix.

DURATION:
-preincubation period (experimentII): 30 °C, for 30 min
- exposure duration: 48 h at 37 °C in the dark

NUMBER OF REPLICATIONS: 3 plates per strain and dose level including the controls
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, WP2 uvrA) or thrice (strains TA 1535, TA 1537) the colony count of the corresponding solvent colony is observed.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the thresshold is regarded as an indication of mutagenic potential if reproduced in an independent second experiment. however, whenever the colony counts remain within the historical range of negative annd solvent controls such as an increase is not considered biologically relevant.
Statistics:
Arithmetic mean, standard deviation and the ratio of treated versus solvent were calculated
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: occurred at concentrations of 333 µg/plate and more (Experiment I and Experiment II with SP mix), or 1000 µg/plate and more (Experiment II without S9 mix). Nevertheless undissolved particles had no influence on the data recording.

COMPARISON WITH HISTORICAL CONTROL DATA:
The laboratory´s historical control data was slightly exceeded in the neative control of strain WP2 uvrA in the presence of metabolic activation in exp. I. This effect is judged to be based on biologically irrelevant fluctuations in the number of colonies and has no impact on the outcom of the study.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

The test item did not exert mutagenic activity in the reverse bacterial mutation assay (plate incorporation assay and pre-incubation assay according to Prival) with and without metabolic activation.
Executive summary:

Mutagenic activity of the test item was investigated in Salmonella typhimurium strains TA 1535, TA 1537, TA98 and TA100 as well as Escherichia coli strain WP2 uvrA with (induced rat liver S9 mix) and without metabolic activation at concentrations of 0, 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate using the plate incorporation assay.

Due to the test items characteristic as an azo-dye the test was also conducted using the Prival modification, i.e. testing the above mentioned bacterial strains in the preincubation assay without and with uninduced hamster liver S9 mix for metabolic activation. This test was performed using the following concentrations: 0, 33, 100, 333, 1000, 2500, 5000 µg/plate.

The test item did not reveal any mutagenic activity under the conditions tested. The appropriate reference mutagenes showed distinct positive mutagenic effects.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

No Classification, as no adverse effects observed.