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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Under the experimental conditions reported, including the Prival test method modification for azo compounds, the substance (Pigment Red 112) did not induce gene mutations by frameshifts or base-pair substitutions in the genome of the bacterial strains used. Therefore, the Pigment Red 112 is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay. Several further mutagenicity tests in bacteria revealed also negative results for Pigment Red 112

Pigment Red 112 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 an in-vitro micronucleus assay in Chinese hamster V79 cells, Pigment Red 112 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

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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:
1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test performed according to OECD test guideline and GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
additionally modified version for azo-dyes
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Principles of method if other than guideline:
The assay was performed with and without rat liver and hamster liver microsomal activation.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
TA 1537: hisC3076, rfa, uvrB
TA 98: hisD3052, rfa, uvrB +R
TA 1535: hisG46, rfa, uvrB
TA 100: hisG46, rfa, uvrB +R
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from rat liver (10%) and S9 mix from hamster liver (30%)
Test concentrations with justification for top dose:
Experiment I: 0, 4, 20, 100, 500, 2500 and 5000 µg/plate;
Experiment II and toxicity test: 0, 4, 20, 100, 500, 2500 and 5000 µg/plate
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Without metabolica activation: sodium azide (TA100, TA1535), 9-aminoacridine (TA1537), 2-nitrofluorene (TA98); with rat liver S9-mix: 2-aminoanthracene (all strains); with hamster liver S9-mix: 2-aminoanthracene (TA100, TA1535, TA1537); congo red (TA98)
Details on test system and experimental conditions:
The assay was performed in two independent experiments:

experiment I: plate incorporation assay with and without induced rat liver S9 mix
experiment II: pre-incubation test with and without non-induced hamster liver S9 mix

Two independent experiments for each of the two protocols (incorporation and pre-incubation) were performed.

Hamster liver S9 mix, but not rat liver S9 mix, contained the reductive agent FMN.


DURATION
- Preincubation period: 30 min, 30°C
- Exposure duration: after solidification the plates were incubated for about 48 hours at 37°C in the dark

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY: existence of evaluable plates (> 0 colonies) at five concentrations or more
Evaluation criteria:
A test item is considered as a mutagen if
a) it produces at least a 2-fold increase in the mean number of
revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate control at complete bacterial lawn, or
b) it induces a dose dependent increase in the mean number of revertants per plate of at least
one of the tester strains over the mean number of revertants per plate of the appropriate vehicle
control in at least two or three concentrations of the test compound at complete bacterial background lawn.
Statistics:
not required
Key result
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
Remarks:
Visible precipitation of the test compound was observed at concentrations of 500 µg/plate and higher
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
Interpretation of results (migrated information): negative with and without metabolic activation

Under the experimental conditions reported, including the Prival test method modification for azo compounds, the test item did not induce gene mutations by frameshifts or base-pair substitutions in the genome of the strains used. Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.
Executive summary:

Under the experimental conditions reported, including the Prival test method modification for azo compounds,  the test item did not induce gene mutations by frameshifts or base-pair substitutions in the genome of the strains used. Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

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
Key result
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

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.
Executive summary:

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 / 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:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
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:
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:
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
Remarks:
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.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
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.

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 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

Pigment Red 112 does not have to be classified for mutagenicity according to the criteria laid down in the EU Classification Labelling and Packaging Regulation (1272/2008/EC) because Pigment Red 112 did not reveal any mutagenic effect in several bacterial reverse mutation assays in the presence or absence of metabolic activation at concentrations up to 5000 µg/plate, in the mammalian cell gene (HPRT) mutation test in V79 Chinese Hamster cells at concentrations up to 400 µg/ml and in the in vitro micronucleus test in V79 cells at up to a precipitating concentration of 31.3 µg/ml.