Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item Pigment Yellow 180 showed no mutagenic activity in a bacterial mutagenicity assay (Ames) in both experiments (plate incorporation assay, preincubation assay) each with and without metabolic activation in five strains.

The analogue substance Pigment Orange 36 did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test substance is considered to be non-mutagenic in this HPRT assay.

The analogue substance Pigment Yellow 175 did not increase the mutant frequency at the TK+/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the Mouse Lymphoma test.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From2 SEP 2004 to 13 SEP 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD TG 471)
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, 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 (induced with phenobarbital/beta-naphtoflavone; experiment I); hamster liver S9 (non-induced; experiment II)
Test concentrations with justification for top dose:
Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate.
Experiment II: 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility properties of the solvent 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 (for all strains)
Remarks:
with metabolic activation (rat liver S9)
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, WP2 uvrA), congo red (TA 98)
Remarks:
with metabolic activation (hamster liver S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Experiment I: plate incorporation assay without and with induced rat liver S9 mix (induction with phenobarbital/beta-naphthoflavone)
Experiment II: preincubation assay without and with non-induced hamster liver S9 mix

DURATION
- Preincubation period: Experiment II: 30° C for 30 minutes
- Exposure duration: at least 48 hours at 37° C

NUMBER OF REPLICATIONS: 3 plates per strain and dose level, including the control





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 threshold 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 an increase is not considered biologically relevant.
Statistics:
Arithmetic means and standard deviation of the counted colonies 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:
cytotoxicity
Remarks:
strain TA1535, TA1537 (exp.I, w/o S9, from 2500 µg/plate), in TA98 (exp. I, w/o S9, from 1000 µg/plate), in TA100 (exp.I, w/o S9, at 5000 µg/plate), in TA1537, TA 98 (exp.I, with S9, from 2500 µg/plate), in TA1535, TA98 (exp.II, w/o S9, at 5000 µg/plate)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
in experiment I w/o S9 mix at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
The test item precipitated in the overlay agar at 333 to 5000 µg/plate in experiment I and at 1000 to 5000 µg/plate in experiment II with and without metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA:
In strain TA 1535 (exp. I), TA 1537 (exp. I and II), TA 100 (exp. I), and WP2 uvrA (exp. I and II) with metabolic activation the historical range of positive controls was not reached. This minor effect was judged to represent fluctuations. The threshold of three times ( TA 1535, TA 1537) and two times (TA 100 and WP2 uvrA) the corresponding solvent control was exceeded, so the test was considered valid.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Toxic effects, evident as a reduction in the number of revertants, were observed at the following concentrations (µg/plate).

Strain

Experiment I

Experiment II

 

without S9 mix

with S9 mix

without S9 mix

with S9 mix

TA 1535

2500, 5000

/

5000

/

TA 1537

2500, 5000

2500, 5000

/

/

TA 98

1000 - 5000

2500, 5000

5000

/

TA 100

5000

/

/

/

WP2 uvrA

5000

/

/

/

/ = no toxic effects observed

The test item precipitated in the overlay agar at the following concentrations (µg/plate):

Strain

 

Experiment I

 

 

Experiment II

 

 

without S9 mix

with S9 mix

without S9 mix

with S9 mix

TA 1535

333

- 5000

333

- 5000

1000

- 5000

1000

- 5000

TA 1537

333

- 5000

333

- 5000

1000

- 5000

1000

- 5000

TA 98

333

- 5000

333

- 5000

1000

- 5000

1000

- 5000

TA 100

333

- 5000

333

- 5000

1000

- 5000

1000

- 5000

WP2 uvrA

333

- 5000

333

- 5000

1000

- 5000

1000

- 5000

The undissolved particles had no influence on the data recording.

Conclusions:
Interpretation of results (migrated information):
negative

The test item showed no mutagenic activity in both experiments (plate incorporation assay, preincubation assay) each 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 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 concentrations 10, 33, 100, 333, 1000, 2500 and 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:
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:
From 2 NOV 2004 to 27 DEC 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD TG 476)
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media:
RPMI 1640 (Hepes buffered medium) supplemented with 15% horse serum, penicillin/streptomycin (100 U/mL respectively 100 µl/mL), 220 µg/mL sodium pyruvate and 1.25 U/mL Amphotericin
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 (induced with phenobarbital/beta-naphtoflavone)
Test concentrations with justification for top dose:
Experiment I:
without S9 mix: 11.7; 23.4; 46.9; 93.8; 187.5, and 375.0 microgram/mL
with S9 mix: 23.4; 46.9; 93.8; 187.5, and 375.0 microgram/mL
Experiment II: without S9 mix: 11.7; 23.4; 46.9; 93.8; and 187.5 microgram/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility properties of the solvent and its relative non-toxicity to the cell cultures
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in suspension
DURATION
- Exposure duration: 4 h (Experiment I: with and w/o metabolic activation) and 24 h (Experiment II w/o metabolic activation)
- Expression time (cells in growth medium): 3 days total (i.e. experiment I: 4 hours treatment period plus 68 hours expression time; experiment II: 24 hours treatment period plus 48 hours expression time)
- Selection time (if incubation with a selection agent): 10 to 15 days

SELECTION AGENT (mutation assays): trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: two independent experiments, using two parallel cultures each

NUMBER OF CELLS USED: Per culture 1 x 10EXP7 cells

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth; other: relative suspension growth
Evaluation criteria:
A test item is classified as positive if it induces either a reproducible concentration-related increase in the mutant frequency or a reproducible positive response for at least one of the test points.
A test item producing neither a reproducible concentration-related increase in the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A significant response is described as follows:
The test item is classified as mutagenic if it reproducibly induces a mutation frequency that is at least two times higher than the spontaneous mutation frequency (negative or solvent control) in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase in the mutation frequency. Such an evaluation may be considered independently of the enhancement factor for induced mutants.
However, in the evaluation of the test results the historical variability of the mutation rates in negative and solvent controls and the mutation rates of all negative and solvent controls of this study are taken into consideration.
Results of test groups are rejected if the relative total growth, the relative suspension growth and/or cloning efficiency 1 (survival of the cells after treatment) is less than 10 % of the solvent control or the cloning efficiency 2 (viability) after the expression period is less than 20 %.
Whenever a test item is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used to differentiate point mutations from clastogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and dose dependent shift in the ratio of small versus large colonies clastogenic effects are indicated.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
indicated by reduced relative cloning efficiency 1 in experiment I at 375 µg/mL in both cultures
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
experiment I: 1st culture at 375 µg/mL, 2nd culture at 187.5 µg/mL and above; experiment II: both cultures at 187.5 µg/mL and above
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
Precipitation of the test item was observed by the unaided eye at 187.5 and 375.0 microgram/mL in all experimental parts.

COMPARISON WITH HISTORICAL CONTROL DATA:
The mutation frequency did not exceed the historical range of negative and solvent controls at any concentration with and without metabolic activation. The threshold of two times the number of mutant colonies/10EXP6 cells was reached at 46.9 microgram/mL in the second culture of experiment I (with S9 mix). Since this effect was not observed at higher concentrations or in the parallel culture, this isolated effect was judged as biologically irrelevant.
The mutation frequency of the positive control in the first culture of experiment I fell just short of the historical range of positive controls with 233 versus 292 colonies per 10EXP6 cells. Since the threshold of twice the mutation frequency of the solvent control was clearly exceeded and the mean value of both positive controls is well within the historical range, this effect was considered as irrelevant fluctuation.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In experiment I without metabolic activation relevant toxicity (relative cloning efficiency 1 and / or relative total growth of less than 50 % survival) was detected at 187.5 µg/mL and above in the second culture. In culture I a clear toxic effect occurred at the highest concentration of 375 µg/mL.
In experiment I with metabolic activation a toxic effect indicated by a reduced relative cloning efficiency 1 was observed at 375 µg/mL in both cultures.
In the second experiment (24 h treatment without metabolic activation) relevant toxic effects occurred at 187.5 µg/mL and above in both cultures.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results:
negative

The test substance did not increase the mutant frequency at the TK+/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test.
Executive summary:

Mouse Lymphoma cells (L5178Y TK+/-) were treated with the test substance at five or six concentrations in the range of 11.7 to 375 microgram/mL. Precipitation of the test substance was detectable at dose level 187.5 and 375 microgram/mL. The solvent control gave mutant frequencies within the normal range. The positive controls gave marked increases in the mutant frequency, both in the presence or absence of metabolic activation, indicating the satisfactory performance of the test system. The test substance demonstrated no statistically significant or dose related increases in mutant frequency at any dose level, either with or without metabolic activation, thus indicating that the test item is not mutagenic under the conditions tested.

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:
From 01 JUN 2011 to 09 AUG 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: according to OECD 476 and GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
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:
Phenobarbital/Beta-Naphtoflavone induced Rat liver S9
Test concentrations with justification for top dose:
Experiment I:
without metabolic activation: 9.4, 18.8, 37.5, 75.0, 150, 1200 µg/mL
with metabolic activation: 9.4, 18.8, 37.5, 75.0, 150, 1200 µg/mL

Experiment II:
without metabolic activation: 6.3, 12.5, 25.0, 50.0, 100, 200, 1200 µg/mL
with metabolic activation. 3.1, 6.3, 12.5, 25.0, 50.0, 100 µg/mL
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

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

SELECTION AGENT (mutation assays): 6-Thioguanine


NUMBER OF REPLICATIONS: 2


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


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:
A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
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 non-muta¬genic in this system.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation fre¬quency at least at one of the concen¬trations 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.
However, in a case by case evaluation this decision depends on the level of the correspon¬ding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent con-trols within all experiments of this study was also taken into consideration.
Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: no, but tested up to and including precipitating concentrations
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: Not increased
- Effects of osmolality: Not effected
- Evaporation from medium: Not examined
- Precipitation:
In the range finding pre-experiment precipitation occurred at 18.8 µg/mL and above with metabolic activation. Without metabolic activation precipitation was noted at 37.5 µg/mL and above following 4 and 24 hours treatment.
- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES:
The highest concentration used in the pre-test was 1200 µg/mL limited by the solubility of the test item in DMSO and aqueous medium. Test item concentrations between 9.4 µg/mL and 1200 µg/mL were used to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24 hours treatment) of metabolic activation. No relevant toxic effect occurred up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment.
The test medium was checked for precipitation at the end of each treatment period (4 or 24 hours) prior to removal to the test item. Precipitation occurred at 18.8 µg/mL and above with metabolic activation. Without metabolic activation precipitation was noted at 37.5 µg/mL and above following 4 and 24 hours treatment.
There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item.
Based on the results of the pre-experiment, the individual concentrations of the main experiments were selected. A series of concentrations spaced by a factor of 2 was placed from the soluble up into the lower precipitating range. An additional large step up to the maximum possible concentration of 1200 µg/mL was added in the first experiment with and without metabolic activation and in the second experiment without metabolic activa-tion. In the second experiment with metabolic activation the maximum possible concentra-tion was not again applied in favour of more soluble concentrations in the lower range.

Doses applied in the gene mutation assay with the test substance:

Experiment I / 4 hours treatment concentration in µg/mL
without metabolic activation 9.4 18.8 37.5P 75.0 P 150.0 P 1200.0 P
with metabolic activation 9.4 18.8 P 37.5 P 75.0 P 150.0 P 1200.0 P
Experiment II / 24 hours treatment concentration in µg/mL
without metabolic activation 6.3 12.5 25.0 P 50.0 P 100.0 P 200.0 P 1200.0 P
Experiment II / 4 hours treatment
with metabolic activation 3.1 6.3 12.5 P 25.0 P 50.0 P 100.0 P

P = precipitation

In the first experiment the cultures at the lowest concentration of 9.4 µg/mL with and without metabolic activation were not continued since a minimum of only four analysable concentrations is required by the guidelines. In the second experiment the cultures at 50 and 100 µg/mL without metabolic activation and at 100 µg/mL with metabolic activation were not continued to avoid analysis of too many precipitating concentrations.


COMPARISON WITH HISTORICAL CONTROL DATA: Complies


ADDITIONAL INFORMATION ON CYTOTOXICITY: None
Remarks on result:
other: all strains/cell types tested
Summary Table
  relative relative relative mutant   relative relative relative mutant  
conc. P S9 cloning cell cloning colonies/ induction cloning cell cloning colonies/ induction
µg/mL mix efficiency I density efficiency II 106cells factor efficiency I density efficiency II 106cells factor
        % % %     % % %    
Column 1 2 3 4 5 6 7 8 9 10 11 12 13
Experiment I / 4 h treatment     culture I          culture II
Solvent control with DMSO - 100.0 100.0 100.0 13.3 1.0 100.0 100.0 100.0 18.9 1.0
Positive control (EMS) 150.0 - 108.7 116.7 77.5 116.8 8.8 91.8 80.0 82.7 101.2 5.4
Test item 9.4 - 102.9 culture was not continued# 90.9 culture was not continued#
Test item 18.8 - 103.0 78.0 91.3 14.2 1.1 96.4 99.2 73.8 19.1 1.0
Test item 37.5 P - 90.6 106.2 92.0 18.5 1.4 88.7 112.8 112.8 10.1 0.5
Test item 75.0 P - 99.4 85.7 83.7 12.3 0.9 93.5 103.3 78.6 40.2 2.1
Test item 150.0 P - 91.1 112.1 76.2 22.0 1.7 75.9 87.6 73.7 20.6 1.1
Test item 1200.0 P - 97.1 82.2 90.5 20.0 1.5 88.6 112.8 70.2 25.8 1.4
Solvent control with DMSO + 100.0 100.0 100.0 14.4 1.0 100.0 100.0 100.0 18.8 1.0
Positive control (DMBA) 1.1 + 102.4 92.8 58.0 1320.1 91.7 105.9 91.9 78.4 919.6 49.0
Test item 9.4 + 99.9 culture was not continued# 95.8 culture was not continued#
Test item 18.8 P + 98.5 78.0 83.2 16.4 1.1 105.3 92.9 92.1 11.7 0.6
Test item 37.5 P + 105.6 91.1 106.4 6.9 0.5 100.4 91.8 111.5 12.9 0.7
Test item 75.0 P + 99.4 101.3 75.7 15.6 1.1 93.1 104.6 88.6 22.4 1.2
Test item 150.0 P + 97.9 80.7 112.0 10.3 0.7 94.0 98.8 80.7 11.9 0.6
Test item 1200.0 P + 99.4 87.8 79.2 23.5 1.6 93.8 85.3 80.5 21.2 1.1
Experiment II / 24 h treatment     culture I          culture II
Solvent control with DMSO   - 100.0 100.0 100.0 18.2 1.0 100.0 100.0 100.0 17.5 1.0
Positive control (EMS) 150.0 - 92.1 90.3 71.5 244.9 13.5 66.2 87.1 89.8 334.0 19.0
Test item 6.3 - 98.3 86.9 100.6 6.1 0.3 83.3 106.8 110.1 11.5 0.7
Test item 12.5 - 99.9 98.8 108.0 10.3 0.6 88.0 111.0 112.7 13.2 0.8
Test item 25.0 P - 99.4 81.7 99.1 14.4 0.8 83.4 99.6 92.3 17.0 1.0
Test item 50.0 P - 100.6 culture was not continued## 92.5 culture was not continued##
Test item 100.0 P - 99.6 culture was not continued## 71.1 culture was not continued##
Test item 200.0 P - 100.1 76.4 99.5 9.8 0.5 72.1 99.1 100.7 16.3 0.9
Test item 1200.0 P - 95.8 81.3 95.5 23.3 1.3 66.3 93.8 91.9 15.2 0.9
Experiment II / 4 h treatment        
Solvent control with DMSO   + 100.0 100.0 100.0 18.1 1.0 100.0 100.0 100.0 16.2 1.0
Positive control (DMBA) 1.1 + 42.2 55.7 104.1 815.4 45.0 51.5 62.7 77.9 1004.5 61.8
Test item 3.1 + 94.4 82.4 124.1 15.8 0.9 102.4 84.7 90.3 11.0 0.7
Test item 6.3 + 95.1 74.8 130.1 14.4 0.8 101.3 107.0 95.1 14.7 0.9
Test item 12.5 P + 96.0 78.8 128.6 15.0 0.8 103.2 81.8 96.4 15.0 0.9
Test item 25.0 P + 93.2 94.1 138.4 13.5 0.7 102.1 81.3 94.4 13.6 0.8
Test item 50.0 P + 95.6 84.8 131.2 26.4 1.5 102.1 94.8 95.0 23.3 1.4
Test item 100.0 P + 97.3 culture was not continued## 102.3 culture was not continued##

#    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

Conclusions:
Interpretation of results (migrated information):
negative

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test substance is considered to be non-mutagenic in this HPRT assay.

Executive summary:

The test item was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster.

The study was performed in two independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

The highest applied concentration of 1200 µg/mL was limited by the solubility properties of the test item in DMSO and aqueous medium.

No relevant cytotoxic effects defined as a reduction of the relative cloning efficiency I and/or relative cell density to values below 50% in both parallel cultures were noted up to the maximum concentration with and without metabolic activation.

No relevant and reproducible increase in mutant colony numbers/106cells was observed in the main experiments up to the maximum concentration. The mutation frequency remained within the historical range of solvent controls.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. No significant dose dependent trend of the mutation frequency indicated by a probabilityvalue of <0.05 was determined in any of the experimental groups.

In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 13.3 up to 18.9 mutants per 106cells; the range of the groups treated with the test item was from 6.1 up to 40.2 mutant colonies per 106cells.

EMS (150 µg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

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

Genetic toxicity in vivo

Description of key information

The analogue substance Pigment Orange 36 did not induce micronuclei in erythrocytes in mice in vivo. Therefore, the test item is considered to be mutagenic.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 25 MAR 1981 to 2 APR 1981
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Basic data given: comparable to guidelines
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Hoechst AG, company breeding colony
- Age at study initiation: 7 to 12 weeks
- Weight at study initiation: males mean: 33 g; females mean: 26 g
- Housing: grouped (5 animals per cage) in macrolon cages (type 3) in fully airconditioned rooms
- Diet: rats/mice diet Altromin1324 (Altromin GmbH, Lage/Lippe, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-2
- Humidity (%): 55+/-10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: 2% starch mucilage
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test compound:
Preparation of compounds dilution was done freshly each day.
6250 mg of test item were weight in a beaker, mixed with 2% starch mucilage, transferred into a 25 mL flask and topped up to the calibration mark. A solution was formed by stirring for 5 minutes on a magnetic stirrer.

Positive control:
For Endoxan(R) (= Cyclophosphamide) stock solution, 5 ml of distilled water were added to 100 mg Endoxan(R) in an injection phial and shaken to form a clear solution. The solutions for administration were prepared from this stock solution. For this purpose, 2 ml of the 2% stock solution were mixed with 6 ml distilled water.
Duration of treatment / exposure:
30 hours in total (1st application, lack phase of 24 hours, 2nd application, after 6 hours termination of test)
Frequency of treatment:
twice
Post exposure period:
6 hours
Remarks:
Doses / Concentrations:
0, 50, 500, 5000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: oral, gavage
- Doses / concentrations: 100 mg/kg bw
Tissues and cell types examined:
polychromatic erythrocytes derived from the bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
According to a preliminary study, the maximal applicable dose of the test item is 5000 mg per kg bodyweight, which was administered twice.

DETAILS OF SLIDE PREPARATION:
At the indicated time a suspension of the bone marrow of both femora was formed. The mixture was then centrifuged for 5 minutes at about 1000 rpm and almost all the supernatant discarded. One drop of the thoroughly mixed sediment was smeared an a cleaned slide and air-dried for about 24 hours.

Staining procedure
- 3 minutes in May-Grünwalds solution
- 2 minutes in May-Grünwalds solution diluted 1:1 with distilled water
- brief rinsing twice in distilled water
- 10 minutes staining in 1 part Giemsa solution to 6 parts distilled water
- rinsing in distilled water
- drying with filter paper
- cleaning the backside of the slide with methanol
- 5 minutes in xylene
- coating with Entellan
Evaluation criteria:
2000 polychromatic erythrocytes were counted for each animal. The number of cells with micronuclei was recorded, not the number of individual micronuclei. As a control measure 1000 mature erythrocytes were also counted and examined for micronuclei. In addition, the ratio of polychromatic to normochromatic erythrocytes was determined. All bone marrow smears for evaluation are coded to ensure that the group to which they belonged remains unknown to the investigator.
Statistics:
The number of polychromatic erythrocytes with micronuclei occurring in the 2000 polychromatic erythrocytes counted, and the number of normocytes with micronuclei occurring in the 1000 normocytes counted, were evaluated statistically. Increases in dose groups compared to the simultaneous control group were determined using binominal distribution. Differences in dose groups compared to the simultaneous control group were checked using the method of Nemenyi (separately for both sexes).
The statistical evaluations were performed using a in-house computer program. All statistical results are based on a 95 % level of significance.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Faeces was stained orange in all animals of dose group 500 and 5000 mg/kg bw.
Conclusions:
Interpretation of results (migrated information): negative
The results indicate that, under the conditions of the present study, the test item is not mutagenic in the micronucleus test.
Executive summary:

The test item was tested in a micronucleus test conducted similar to OECD guideline 474. The test compound was administered orally by gavage to male and female mice. The following doses were tested: 0, 50, 500 and 5000 mg per kg bodyweight.

The animals were treated twice within 24 h with the test compound and according to the test procedure the animals were killed 6 hours after the second administration of the test compound.

The incidence of micronucleated polychromatic erythrocytes of the animals treated with the test item was within the normal range of the negative control. The number of normochromatic erythrocytes containing micronuclei was not increased compared to the control animals.The ratio of polychromatic/normochromatic erythrocytes in both male and female animals remained unaffected by the treatment.

The positive control (Cyclophosphamide = EndoxanR) yielded positive results and therefore indicating the sensitivity of the system.

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

Additional information

Justification for classification or non-classification

No classification: No adverse effects observed