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Genetic toxicity in vitro

Description of key information

Pigment Red 57:1(Ca) is not mutagenic in bacteria as assessed both with standard Ames tests and the modified Prival assay (MHLW 1993). It is also not mutagenic in mammalian cells in vitro (Wollny 2008). It is not clastogenic in vitro (Hoffmann 2008 and MHLW 1993) and it does not induce unscheduled DNA synthesis in human fibroblasts and primary hepatocytes from arochlor 1253-induced rats (Hertner 1987 and Meyer 1987). Pigment Red 57:1(Ca) is therefore considered to be non-genotoxic in vitro.

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Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
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 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
- Stability in Solvent: Not indicated by the sponsor
- Storage: Room temperature
- Expiration Date: October 31, 2017 (Statement of producer)
- Identity: Graphtol-Rubine 6BPB
- Batch No.: KRON 792014
- Composition: C.I. Pigment Red 57:1, 84.34 % (w/w); C.I. Pigment Red 63:1, 7.73 % (w/w)
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 into 15 mL of MEM (Minimal Essential Medium; Seromed, 12247 Berlin, Germany) supplemented with 10 % fetal calf serum (FCS; PAA Laboratories GmbH, 35091 Cölbe, 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 sub-cultured twice weekly. The cell cultures were incubated at
37 °C in a humidified atmosphere with 1.5 % carbon dioxide (98.5 % 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: 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000 µg/mL
Exp II without S9 mix: 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000 µg/mL
Exp II with S9 mix: 15.6, 31.3, 62.5, 125, 250, 500 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: On the day of the experiment (immediately before treatment), the test item was suspended in DMSO (E. MERCK, 64293 Darmstadt, Germany; purity 99.5 %). The final concentration of DMSO in the culture medium was 0.5 % (v/v). The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
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 4 hours with S9 mix and 18 hours without S9 mix. The chromosomes were prepared 18 hours after start of treatment with the test item.

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk


DURATION
- Exposure duration: 4 and 18 hours
- Expression time (cells in growth medium): 18 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 18 hours

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

NUMBER OF REPLICATIONS: 1.5

NUMBER OF CELLS EVALUATED: 200

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; relative total growth

OTHER EXAMINATIONS:
- Determination of polyploidy: no evidence of an increase in the rate of polyploid cells
- Determination of endoreplication: no evidence of an increase in the rate of endomitotic cells

Evaluation criteria:
Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik") using NIKON microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. 100 well spread metaphases per culture were scored for cytogenetic damage on coded slides.
Only metaphases with characteristic chromosome numbers of 22 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined.
Statistics:
Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05).
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The test item Graphtol-Rubine 6BP, suspended in DMSO, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in the absence and the 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 4 hours with S9 mix and 18 hours without S9 mix. The chromosomes were prepared 18 hours after start of treatment with the test item.

In each experimental group two parallel cultures were set up. 100 metaphases per culture were scored for structural chromosome aberrations. No relevant influence of the test item on the osmolarity and the pH value was observed (Exp. I: solvent control 330 mOsm, pH 7.3 versus 370 mOsm, pH 7.3 at 1000.0 µg/mL; Exp. II: solvent control 393 mOsm, pH 7.4 versus 376 mOsm, pH 7.4 at 1000.0 µg/mL).

Test item precipitation was observed starting at a concentration of 250.0 µg/mL in Experiment I as well as in Experiment II in the absence of S9 mix and starting at a concentration of 125.0 µg/mL in Eperiment II in the presence of S9 mix. No cytotoxic effects indicated by reduced cell numbers and/or mitotic indices of below 50 % of control were observed in all experimental parts. In both experiments in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed . The aberration rates of the cells after treatment with the test item (0.0 - 2.0 % aberrant cells excluding gaps) were close to solvent control values (1.5 - 2.5 % aberrant cells excluding gaps) and lay within the laboratory’s historical control data range (0.0 – 4.0 % aberrant cells excluding gaps).

No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to controls. In both experiments, either EMS (500.0 or 900.0 µg/mL) or CPA (1.4 µg/mL) were used as positive controls and showed distinct increases in the number of cells with structural chromosome aberrations. In conclusion, it can be stated that under the experimental conditions reported, the test item Graphtol-Rubine 6BP did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line) when tested up to precipitating concentrations.

Summary of results of the chromosome aberration study with Graphtol-Rubine 6BP

Exp.

Preparation

Test item

Cell numbers

Mitotic indices

Aberrant cells

 

interval

concentration

in %

in %

in %

 

in µg/mL

of control

of control

incl. gaps*

excl. gaps*

with exchanges

 

 

Exposure period 4 hrs without S9 mix

I

18 hrs

Solvent control1

100.0

100.0

2.5

1.5

0.0

 

Positive control2

n.t.

83.9

12.0

11.5S

5.5

 

62.5

94.6

90.4

1.5

1.0

0.0

 

125.0

93.6

98.1

1.5

1.5

0.5

 

250.0P

108.6

100.3

2.5

2.0

0.5

 

 

Exposure period 18 hrs without S9 mix

II

18 hrs

Solvent control1

100.0

100.0

2.0

1.5

0.5

 

Positive control3

n.t.

90.0

25.0

23.0S

3.0

 

62.5

124.1

93.3

0.5

0.0

0.0

 

125.0

83.5

97.1

1.5

1.0

0.0

 

250.0P

93.1

65.1

0.5

0.5

0.0

 

 

Exposure period 4 hrs with S9 mix

I

18 hrs

Solvent control1

100.0

100.0

1.5

1.5

0.5

 

Positive control4

n.t.

74.4

12.0

10.0S

0.5

 

62.5

77.7

63.1

1.0

1.0

0.5

 

125.0

98.9

109.7

0.5

0.5

0.0

 

250.0P

82.1

90.9

2.5

2.0

0.5

 

II

18 hrs

Solvent control1

100

100

3.0

2.5

0.0

 

Positive control4

n.t.

69.3

13.0

11.0S

4.5

 

31.3

94.8

93.4

1.5

1.5

0.5

 

62.5

91.1

113.6

1.0

0.5

0.5

 

125.0P

79.8

102.1

2.0

2.0

0.0

 

*      Inclusive cells carrying exchanges

n.t.  Not tested

P       Precipitation occurred

S      Aberration frequency statistically significant higher than corresponding control values

1      DMSO  0.5 % (v/v)

2            EMS 900.0 µg/mL

3      EMS 500.0 µg/mL

4            CPA     1.4 µg/mL

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Principles of method if other than guideline:
- First experiment 4 hours treatment with and without metabolic activation
- Second experiment 24 hours treatment without metabolic activation
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: mammalian cell gene mutation assay
Specific details on test material used for the study:
- Physical state: Solid
- Purity test date: Certificate of Analysis AZ 353/e1, dated December 10, 2007
- Expiration date of the lot/batch: October 31, 2017
- Storage condition of test material: Room temperature
- Name of test material (as cited in study report): Graphtol-Rubine 6BP
- Composition of test material: C.I. Pigment Red 57:1; 84.34 % (w/w); C.I. Pigment Red 63:1; 7.73 % (w/w)
- Lot/batch No.: KRON 792014

Target gene:
HPRT
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO;
- Justification for choice of solvent/vehicle: solubility properties
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours with and without metabolic activation in experiment 1, 24 hours without metaoblic activation in experiment 2
- Expression time (cells in growth medium): 72 hours
- Selection time (if incubation with a selection agent): 10 days

SELECTION AGENT (mutation assays): Thioguanine

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: >1,5x10exp.6

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency


Evaluation criteria:
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered to be non-mutagenic in this system. A mutagenic response is described as follows:
- The test item is classified as mutagenic if it induces reproducibly with one of the concen¬trations a mutation frequency that is three times higher than the spontaneous mutation fre¬quency in the experiment.
- The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
- In a case by case evaluation this decision depends on the level of the corresponding solvent control data.
Statistics:
Linear regression analysis (least squares) .
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Precipitation: precipitation occurred at 125 µg/mL and 1000 µg/mL in experiment I with and without metabolic activation (4 h treatment), and at 62.5 µg/mL, 125 µg/mL, and 1000 µg/mL in experiment II without metabolic activation (24 h treatment)

RANGE-FINDING/SCREENING STUDIES: determination of plating efficiency, concentration range 7.8 to 1000 µg/mL.
- Cytotoxicity at 500 µg/mL and above without metabolic activation at 4 h treatment.
- Precipitation at 4 h treatment without metabolic activation: at 125 µg/mL and 1000 µg/mL
- Precipitation at 4 h treatment with metabolic activation: at 62.5 µg/mL, 125 µg/mL, and 1000 µg/mL
- Precipitation at 24 h treatment without metabolic activation: at 125 µg/mL and 1000 µg/mL

COMPARISON WITH HISTORICAL CONTROL DATA: all mutant frequencies within historical control data
Summary Table
      relative relative mutant   relative relative mutant  
  conc. S9 cloning cloning colonies/ induction cloning cloning colonies/ induction
  µg/mL mix efficiency 1 efficiency 2 106 cells factor efficiency 1 efficiency 2 106 cells factor
    % %   % %  
column 1 2 3 4 5 6 7 8 9 10
Experiment I     culture I          culture II
Solvent control DMSO - 100.0 100.0  11.9 1.0 100.0 100.0  15.2 1.0
Pos. contr. with EMS 150.0 -  86.5  76.2  87.2 7.3  82.8  66.8 237.9 15.6
Test item   7.8 - 100.6 culture was not continued#  89.6 culture was not continued#
Test item  15.6 - 103.5  91.9  11.3 0.9  99.0  90.3  26.4 1.7
Test item  31.3 - 103.8  80.2  13.5 1.1  93.2  98.2  16.1 1.1
Test item  62.5 -  97.0  93.8   7.9 0.7  95.1  88.6  19.3 1.3
Test item 125.0 (p) - 101.3  84.1   9.1 0.8  90.6  91.2  24.9 1.6
Test item 1000.0 (p) -  98.0  79.1  17.0 1.4  82.8  87.2  20.6 1.4
       
Solvent control DMSO + 100.0 100.0   5.4 1.0 100.0 100.0  27.5 1.8
Pos. contr. with DMBA   1.1 +  75.1  54.9 898.8 165.3  74.1  61.9 247.0 16.2
Test item   7.8 +  97.8  83.4  19.8 3.6  96.7  81.4   8.8 0.6
Test item  15.6 + 100.8  70.9  18.0 3.3  94.9  86.7   4.3 0.3
Test item  31.3 +  95.7  70.0  13.0 2.4 103.6  79.1   3.9 0.3
Test item 62.5 (p) +  98.7  77.2   9.2 1.7  93.9  86.0  11.4 0.7
Test item 125.0 (p) +  97.0 culture was not continued##  95.7 culture was not continued##
Test item 1000.0 (p) +  88.6  67.7  21.9 4.0  68.0  83.9   7.4 0.5
Experiment II     culture I          culture II
Solvent control DMSO - 100.0 100.0  19.4 1.0 100.0 100.0  20.4   1.0
Pos. contr. with EMS 150.0 -  98.4  43.0 710.5 36.6  82.1  89.3 400.3  19.6
Test item 7.8 -  97.6  83.2  26.8 1.4  86.4 112.7  12.1   0.6
Test item 15.6 -  98.1 105.1   7.7 0.4  84.4 129.4  15.2   0.7
Test item 31.3 -  98.8 107.9  11.6 0.6  95.8 116.8  18.4   0.9
Test item 62.5 (p) -  99.1 107.0  20.3 1.0  84.7  91.5  17.0   0.8
Test item 125.0 (p) -  94.6 culture was not continued##  87.2 culture was not continued##
Test item 1000.0 (p) -  93.6 103.8  13.0 0.7  89.7 109.7  11.1   0.5

#     culture was not continued since a minimum of only four analysable concentrations is required

##   culture was not continued to avoid analysis of too many precipitating concentrations
p     precipitation or turbidity visible to the unaided eye

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Additional Prival Assay performed with two strains
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Purity: approximately 98% [ Containing as impurities : NaCl, CaCl2, Ca(OH)2, and water ]
- Lot/Batch No : T-1322-2
- Storage condition of test material : Stored in cold-dark conditions
- Molecular weight: 424.45
- Stability in the vehicle : Stable for 4 hours in the vehicle at the concentrations of 3 and 50.0 mg/ml, according to the stability test performed by the laboratory.
Target gene:
his, trp
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Species / strain / cell type:
other: TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9, induced with phenobarbital and 5,6-benzophenone
Test concentrations with justification for top dose:
Cytotoxicity test: 50, 150, 500, 1500 and 5000 μg/plate; Genotoxicity test: 312, 625, 1250, 2500 and 5000 μg/plate
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide: TA100, TA98 and E.coli WPA uvrA, without S9; Trypane blue: TA 98 and TA 100 (hamster) S9; 2-aminoanthracene: all strains, with S9
Remarks:
TA100, TA98 and E.coli WPA uvrA without S9
Details on test system and experimental conditions:
Precipitation of the test substance was observed at all doses with exception of 312 and 625 μg per plate at the pre-incubation experiment with S9 of the Prival Assay.
Evaluation criteria:
The test item is judged to be positive when a dose-dependent and reproducible increase in the number of revertant colonies (at least twice as high as that of the spontaneous mutation rate) in at least one tester strain either with or without S9 mix is observed.
Statistics:
not applied
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 nor precipitates, but tested up to recommended limit concentrations
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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
No independent repeat experiment performed
GLP compliance:
yes
Type of assay:
other: in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
- Purity: approximately 98% [ Containing as impurities : NaCl, CaCl2, Ca(OH)2, and water ]
- Lot/Batch No : T-1322-2
- Storage condition of test material : Stored in cold-dark conditions
- Stability in the vehicle: Stable for 4 hours in the vehicle at the concentrations ranging between 0.925 and 50.0 mg/ml, according to the stability test performed by the laboratory.
Species / strain / cell type:
other: Chinese Hamster CHL/IU
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9, induced with phenobarbital and 5,6-benzophenone
Test concentrations with justification for top dose:
Cell growth inhibition test :
Short-term treatment (6 hr-treatment/18 hr-recovery) : 0.16, 0.31, 0.63, 1.25, 2.5, 5.0 mg/L
Continuous treatment (48hr-treatment) : 0.16, 0.31, 0.63, 1.25, 2.5, 5.0 mg/L

Chromosome aberration test :
Continuous treatment (24 and 48h) without S9: 0.09, 0.19 and 0.37 mg/L
short-term treatment (6h/18h recovery) with and without S9: 1.3, 2.5 and 5 mg/L
Vehicle / solvent:
0.5% carboxymethyl cellulose sodium in water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Remarks:
with S9
Details on test system and experimental conditions:
Two independent experiments were performed.
In Experiment I the exposure period was 6 hours with and without metabolic activation. The chromosomes were prepared 18 hours after start of treatment with the test item.
In Experiment II the exposure period was 24 and 48h hours without S9 mix.


METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk


DURATION
- Exposure duration : Short-term treatment test - 6 hours, Continuous treatment test - 24 hours and 48 hours
- Expression time (cells in growth medium): 18 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 18 hours


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


NUMBER OF CELLS EVALUATED: 200 in total per dose for evaluating structural aberrations, 800 in total per dose for evaluating polyploidy


DETERMINATION OF CYTOTOXICITY
- Method: relative total growth


OTHER EXAMINATIONS:
- Determination of polyploidy: no evidence of an increase in the rate of polyploid cells
Evaluation criteria:
The result was considered negative when the occurrence rate of cells with numerical or structural aberrations was below 5 % and false positive for a rate between 5 - 10 %, and positive for above 10%.
Statistics:
Fisher's exact probability test was applied.
Species / strain:
other: Chinese Hamster CHL/IU
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Cytotoxicity as reduction by cell growth was observed in the experiment with continuous exposure only.

Statistically significant increases were noted in the occurrence rate of cells with structural or numerical aberrations in the short-term test at the following concentrations, however, the findings were considered negative with below 5%,  based on the evaluation criteria:

- 1.3 mg/L, Without S9, short-term treatment : No of cells with aberration 2.5% (p = 0.0304)

- 5.0 mg/L, Without S9, short-term treatment : Polyploidy  4.75 % (p =1.97 X 10-8)

- 5.0 mg/L, With S9, short-term treatment : Polyploidy  1.0 % (p =0.0192)

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In vitro mutagenicity in bacteria

The mutagenicity in vitro was assessed in several tests applying both the standard assay with rat liver homogenate and the modified assay for azo compounds (Prival-assay). Many of the studies are screening tests with three (TA 98, TA 100 and TA 1535) or four (plus TA 1537) tester strains performed between 1978 and 1986. Tested samples were commercial products containing varying amounts of additives. With the limitation in the number of tester strains, these studies are reliable and valid in regard to design, positive and negative control and concentrations. Four studies were performed following the latest OECD testing guideline (OECD 471, adopted July 12, 1997) and the principles of GLP and three of them included a Prival assay to take into account reductive metabolism of the azo bond.

 

With one exception, all available studies showed absence of mutagenicity in bacteria. In the one study where a mutagenic effect was observed, this was a less than 3-fold increase in mutant frequency in the absence of metabolic activation in strains WP2 uvrA and TA 98 at concentrations in the precipitating range (2500 and 5000 µg/plate). The strain TA98 was evaluated in all other available studies as well and did not give a mutagenic response in any of them. The strain WP2 uvrA was evaluated in three other studies with concentrations of up to 5000 µg/plate without showing a mutagenic response. It is therefore concluded that the single mutagenic response is either incidental or caused by an unidentified impurity in this specific commercial product. Pigment Red 57:1 itself is concluded to be non-mutagenic in bacteria. The study by MHLW (1993) was chosen as key study because it includes both the standard and the Prival assay and used the sample with the highest content of Pigment Red 57:1.

 

In vitro mutagenicity in mammalian cells

Mutagenicity in mammalian cells in vitro was investigated in an HPRT test following OECD testing guideline 476 (adopted July 21, 1997) and the principles of GLP. No indication of a mutagenic effect was observed at concentrations up to 1000 µg/mL, the two highest concentrations being in the precipitating range. This is consistent with the absence of unscheduled DNA synthesis as tested with both with human fibroblasts (Meyer 1987) and with primary hepatocytes prepared from arochlor1254-induced rats (Hertner 1987). The latter studies were performed under GLP and in combination fulfil the requirements of OECD testing guideline 482 (adopted October 23, 1986). For the DNA-repair assays, concentrations were in similar range and also resulted in precipitation. Higher concentrations were too toxic for scoring. For all three studies, commercial samples of known composition were tested.

 

In vitro clastogenicity in mammalian cells

Clastogenicity in mammalian cells was investigated in two guideline (OECD 473. adopted July 21, 1997) and GLP compliant studies (Hoffmann 2008 and MHLW 1993). The highest evaluable concentrations were chosen based on visual observation of precipitates. No indication of clastogenicity or polyploidy were observed in either study. For both studies, commercial samples of known composition were tested.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for genetic toxicity according to Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008.