Registration Dossier

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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item showed no mutagenic activity in a bacterial reverse mutation assay (plate incorporation assay, preincubation assay) each with and without metabolic activation.


The test substance is considered to be non-clastogenic in a chromosome aberration test in the absence and presence of metabolic activation, when tested up to precipitating or the highest evaluable concentrations.


 


It is concluded that the test item, the close structural analogue C.I.Pigment Yellow 154, does not have the potential to induce gene mutation in CHO-K1 cells at the tested concentrations and under the conditions of testing employed.

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:
From 27 JUL 2011 to 23 AUG 2011
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
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: minimal essential medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital / beta-naphtoflavone induced rat liver S9
Test concentrations with justification for top dose:
With metabolic activation:
Experiment I: 0.4, 1.0, 2.5, 6.1 , 15.4, 38.4, 96.0, 240.0, 600.0 µg/mL
Experiment II: 1.2, 2.4, 4.7, 9.4, 18.8, 37.5, 75.0, 150.0 µg/mL

Without metabolic activation:
Experiment I: 0.4, 1.0, 2.5, 6.1 , 15.4, 38.4, 96.0, 240.0, 600.0 µg/mL
Experiment II: 1.2, 2.4, 4.7, 9.4, 18.8, 37.5, 75.0, 150.0 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility of test item
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:
cyclophosphamide
Remarks:
with metabolic activation
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. Evaluation of two cultures per dose group.
METHOD OF APPLICATION: in culture medium (minimal essential medium)

DURATION
- Exposure duration: 4 hours (+/- S9 mix) and 18 hours (- S9 mix)
- 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: about 1.5


NUMBER OF CELLS EVALUATED: At least 100 well spread metaphases per culture were evaluated for cytogenetic damage on coded slides, except for the positive control in Experiment II without metabolic activation, where only 50 metaphases were evaluated.


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index and cell numbers


OTHER EXAMINATIONS:
- Determination of polyploidy: 500 per culture
- Determination of endoreplication: 500 per culture
Evaluation criteria:
Evaluation of the cultures was performed according to the OECD Guideline using NIKON microscopes with 100x 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. At least 100 well spread metaphases per culture were evaluated for cytogenetic damage on coded slides, except for the positive control in Experiment II without metabolic activation, where only 50 metaphases were evaluated.
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) and relative cell numbers were determined.

In addition, the number of polyploid cells in 500 metaphases per culture was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype). Additionally the number of endomitotic cells scored at the evaluation of polyploid cells was noticed and reported (% endomitotic metaphases).
Statistics:
Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05).
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
The test item, 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 presence of metabolic activation by S9 mix.
Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without S9 mix. In Experiment II the exposure period was 4 hours with S9 mix and 18 hours without S9 mix. The chromosomes were prepared 18 hours (Exp. I & II) after the start of treatment with the test item.
In each experimental group two parallel cultures were set up. At least 100 metaphases per culture were evaluated for structural chromosome aberrations, except for the positive control in Experiment II without metabolic activation, where only 50 metaphases were evaluated.
No relevant influence of the test item on pH value or osmolarity was observed.
Precipitation of the test item in culture medium was observed in Experiment I at preparation interval 18 hours with 96.0 µg/mL and above. In this experiment severe precipitation could be observed on the prepared slides beginning with 38.4 µg/mL in the absence and presence of S9 mix. In Experiment II after 18 hours continuous treatment precipitation occurred with 4.7 µg/mL and above without S9 mix and in the presence of S9 mix with 9.4 µg/mL and above. Severe precipitation on the slides was observed in the absence of S9 mix beginning with 18.8 µg/mL and 37.5 µg/mL in the presence of S9 mix. Evaluation of the mentioned slides was impossible due to interference of the cells by the precipitate.
In this study, neither reduced mitotic indices nor reduced cell numbers could be observed up to the highest evaluated concentrations of the test item, except in Experiment I, in the presence of S9 mix. In this part of the study, the cell numbers were slightly reduced after treatment with 6.1, 15.4 and 38.4 µg/mL (56.4, 72.8 and 76.5 % of control).
In Experiment I, in the absence and presence of S9 mix and in Experiment II in the absence 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 (1.0 - 3.5 % aberrant cells, excluding gaps) were close to the range of the solvent control values (2.0 - 3.5 % aberrant cells, excluding gaps) and within the range of the laboratory historical control data: 0.0 -4.0 % aberrant cells, excluding gaps. In Experiment II in the presence of S9 mix one single value slightly exceeded the range (6 % aberrant cells, excluding gaps) of the laboratory historical solvent control data (0.0 - 4.0 % aberrant cells, excluding gaps). However, this value was not statistically significant and no dose-dependency was observed with the evaluated concentrations. No relevant effect occurred in Experiment I and thus the finding is regarded as being without biological relevance.
In both experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (2.0 - 5.0 %) as compared to the rates of the solvent controls (2.7 - 3.2 %).
A slightly increased number of endomitotic cells was observed in both experiments (0.0 -1.5 %). As these effects were less frequent and not dose dependent they are regarded as biologically irrelevant.
In both experiments, either EMS (1000.0 or 600.0 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.

Summary of results of the chromosome aberration study with the test substance

Exp.

Preparation

Test item

Endomitotic

Polyploid

Cell numbers

Mitotic indices

Aberrant cells

 

interval

concentration

cells

cells

in %

in %

in %

 

 

in µg/mL

in %

in %

of control

of control

incl. gaps*

excl. gaps*

with exchanges

Exposure period 4 hrs without S9 mix

I

18 hrs

Solvent control1

0.1

3.2

100.0

100.0

3.5

3.5

1.0

 

 

Positive control2

n.d.

n.d.

n.d.

116.1

25.0

25.0S

17.5

 

 

2.5

0.0

5.0

108.5

101.6

1.0

1.0

0.5

 

 

6.1

0.0

3.7

123.2

92.5

2.5

2.0

0.0

 

 

15.4

0.0

3.1

100.8

112.9

2.5

2.0

1.5

Exposure period 18 hrs without S9 mix

II

18 hrs

Solvent control1

0.0

3.1

100.0

100.0

3.5

2.5

0.0

 

 

Positive control#3

n.d.

n.d.

n.d.

68.9

52.0

51.0S

15.0

 

 

1.2

0.0

2.9

88.0

112.4

1.5

1.0

0.0

 

 

2.4

0.0

2.8

94.9

111.6

4.0

3.5

0.5

 

 

4.7P

0.0

2.0

95.4

90.0

3.0

2.0

0.0

Exposure period 4 hrs with S9 mix

I

18 hrs

Solvent control1

0.1

3.1

100.0

100.0

2.0

2.0

1.5

 

 

Positive control4

n.d.

n.d.

n.d.

88.5

23.0

22.5S

9.5

 

 

6.1

1.1

3.5

56.4

121.8

2.5

2.5

1.0

 

 

15.4

1.0

2.7

72.8

101.7

1.5

1.5

0.5

 

 

38.4

0.3

2.3

76.5

108.1

2.0

1.0

1.0

II

18 hrs

Solvent control1

0.0

2.7

100

100

4.0

3.5

1.0

 

 

Positive control4

n.d.

n.d.

n.d.

48.6

16.5

15.0S

6.5

 

 

1.2

1.5

4.0

110.6

115.6

2.5

1.5

0.0

 

 

2.4##

0.2

2.6

111.9

103.8

6.5

6.0

1.3

 

 

4.7

0.1

2.9

103.7

93.4

3.5

3.0

1.0

 

 

9.4P

0.2

2.4

100.1

120.5

4.0

4.0

0.5

*     Inclusive cells carrying exchanges

#     Evaluation of 50 metaphases per culture

##    Evaluation of 200 metaphases per culture

n.d. Not determined

P     Precipitation occurred at the end of treatment

S     Aberration frequency statistically significant higher than corresponding control values

1     DMSO    0.5 % (v/v)

2         EMS 1000.0 µg/mL

3         EMS   600.0 µg/mL

4         CPA       1.4 µg/mL

Conclusions:
Interpretation of results: negative

In conclusion, the test substance is considered to be non-clastogenic in this chromosome aberration test in the absence and presence of metabolic activation, when tested up to precipitating or the highest evaluable concentrations.
Executive summary:

The test item, suspended in DMSO, was assessed for its potential to induce structural chromosome aberrations in V79cells of the Chinese hamster in vitro in two independent experiments. The following study design was performed:

 

Without S9 mix

With S9 mix

 

Exp. I

Exp. II

Exp. I

Exp. II

Exposure period

 4 hrs

18 hrs

 4 hrs

 4 hrs

Recovery

14 hrs

-

14 hrs

14 hrs

Preparation interval

18 hrs

18 hrs

18 hrs

18 hrs

In each experimental group two parallel cultures were set up. At least 100 metaphases per culture were evaluated for structural chromosome aberrations, except for the positive control in Experiment II without metabolic activation, where only 50 metaphases were evaluated.

The highest applied concentration (600.0 µg/mL; approx. 1.5 mM) was chosen with regard to the ability to formulate a homogeneous suspension of the test item in an appropriate solvent.

In the evaluable slides neither in the absence nor in the presence of S9 mix the mitotic index and/or cell numbers were reduced indicating no cytotoxicity.

In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item.

No relevant increase in polyploid metaphases was found after treatment with the test item as compared to the frequencies of the control cultures.

Appropriate mutagens were used as positive controls. They induced statistically significantincreases (p < 0.05) in cells with structural chromosome aberrations.

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 1 APR 2005 to 15 APR 2005
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: 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 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 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:
Minor toxic effects, evident as a reduction in the number of revertants, were observed in the presence of metabolic activation in strain TA 1535 at 5000 µg/plate in experiment II.
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:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
COMPARISON WITH HISTORICAL CONTROL DATA:
The laboratory´s historical control range was slightly exceeded in the negative control of strain WP2 uvrA without metabolic activation in experiment II. Since this deviation is rather small, this effect is judged to be based on biologically irrelevant fluctuations in the number of colonies and has no impact on the outcome of the study.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

TEST-SPECIFIC CONFOUNDING FACTORS

- Precipitation:

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

Conclusions:
Interpretation of results: 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 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 bacteria
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:
From 1 APR 2005 to 15 APR 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD TG 471)
Justification for type of information:
See read across justification document in chapter 13
Reason / purpose for cross-reference:
read-across source
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: 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 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 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:
Minor toxic effects, evident as a reduction in the number of revertants, were observed in the presence of metabolic activation in strain TA 1535 at 5000 µg/plate in experiment II.
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:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
COMPARISON WITH HISTORICAL CONTROL DATA:
The laboratory´s historical control range was slightly exceeded in the negative control of strain WP2 uvrA without metabolic activation in experiment II. Since this deviation is rather small, this effect is judged to be based on biologically irrelevant fluctuations in the number of colonies and has no impact on the outcome of the study.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

TEST-SPECIFIC CONFOUNDING FACTORS

- Precipitation:

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

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 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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2020
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: In vitro mammalian cell gene mutation test using the Hprt gene
Target gene:
HPRT
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
Chinese Hamster (Cricetulus griseus) ovary cell line CHO-K1,
(ATCC CCL-61, Lot 4765275)
Metabolic activation:
with and without
Untreated negative controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
Details on test system and experimental conditions:
Cells were grown in tissue culture flasks at 37 ± 1 °C in a humidified carbon dioxide incubator (5 ± 0.2 % CO2 in air)
Rationale for test conditions:
Test approaches currently accepted under the OECD for the assessment of mammalian cell gene mutation involve the use of Chinese Hamster Ovary (CHO) cell line. This cell line has been demonstrated to be sensitive to the mutagenic activity of a variety of chemicals.

Established CHO cell line is useful in in vitro gene mutation testing because it is easily cultured in standard medium, has a small number of large chromosomes each with a more or less distinctive morphology and a relatively short cycle time
Evaluation criteria:
CRITERIA FOR ACCEPTABILITY OF THE TEST
The assay will be considered valid if the following criteria are met:
a) The concurrent vehicle control data is within the range of the laboratory historical control data.
b) The concurrent positive control substances should induce responses that are compatible with those generated in the historical positive control data base and produce a statistically significant increase compared with the concurrent vehicle control.
c) Two experimental conditions are tested unless one results in positive response.
d) Adequate number of cells and analyzable concentrations are tested under each of the experimental conditions.
e) The criteria for the selection of top concentration are consistent with those described in the guideline.

EVALUATION AND INTERPRETATION OF RESULTS
When all the validity criteria are fulfilled:

1. A test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
• At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent vehicle control
• The increase is concentration-dependent when evaluated with an appropriate trend test
• Any of the results are outside the distribution of the historical vehicle control data

When all of these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.

2 A test chemical is considered to be clearly negative if, in all experimental conditions examined:

• None of the test concentrations exhibits a statistically significant increase compared with the concurrent vehicle control
• There is no concentration-related increase when evaluated with an appropriate trend test
• All results are inside the distribution of the historical vehicle control data

The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Conclusions:
It is concluded that the test item, C.I.Pigment Yellow 194 does not have the potential to induce gene mutation in CHO-K1 cells at the tested concentrations and under the conditions of testing employed.
Executive summary:

 


The genotoxic potential of the test item C.I.Pigment Yellow 194 induce gene mutation in mammalian cells was evaluated using Chinese Hamster ovary (CHO) cells.


 


The study consisted of a preliminary cytotoxicity test and a definitive gene mutation test. The gene mutation test comprised of two independent experiments, one each in the presence and absence of metabolic activation system (S9 fraction prepared from Aroclor 1254 induced rat liver).


 


C.I.Pigment Yellow 194 was insoluble in sterile water and formed a free flowing suspension in Dimethyl sulfoxide (DMSO) at
200 mg/mL.


 


In a preliminary cytotoxicity test for the selection of test concentrations for the gene mutation assay, the Relative Survival was 27 and 32 % at the
1000 µg/mL, in the presence and absence of metabolic activation, respectively. There was precipitation of the test item in the test medium at and above
1000 µg/mL, both in the presence and absence of metabolic activation. There was no appreciable change in the pH and osmolality of test medium. Based on these observations a maximum of 1500 µg/mL was tested in the gene mutation assay.
 


 


In the gene mutation test, CHO-K1 cells were exposed to the test item in duplicate at concentrations of 23.44, 93.75, 375 and 1500 µg/mL of the medium for 3 hours in the presence (Experiment 1) and absence (Experiment 2) of metabolic activation. In a similar way, a concurrent vehicle control (DMSO) and a positive control, 3-methylcholanthrene (Experiment 1) were also tested in duplicate.


 


There was no evidence of induction of gene mutations in any of the test item treated cultures either in the presence or absence of metabolic activation. The positive control in experiment 1 produced a statistically significant increase in the frequencies of mutants, under identical conditions.


 


The results of the forward gene mutation test at thehprtlocus with C.I.Pigment Yellow 194 indicated that the test item was non-mutagenic under the conditions of this study

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:
From 27 JUL 2011 to 23 AUG 2011
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 read across document in 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
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: minimal essential medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital / beta-naphtoflavone induced rat liver S9
Test concentrations with justification for top dose:
With metabolic activation:
Experiment I: 0.4, 1.0, 2.5, 6.1 , 15.4, 38.4, 96.0, 240.0, 600.0 µg/mL
Experiment II: 1.2, 2.4, 4.7, 9.4, 18.8, 37.5, 75.0, 150.0 µg/mL

Without metabolic activation:
Experiment I: 0.4, 1.0, 2.5, 6.1 , 15.4, 38.4, 96.0, 240.0, 600.0 µg/mL
Experiment II: 1.2, 2.4, 4.7, 9.4, 18.8, 37.5, 75.0, 150.0 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility of test item
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:
cyclophosphamide
Remarks:
with metabolic activation
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. Evaluation of two cultures per dose group.
METHOD OF APPLICATION: in culture medium (minimal essential medium)

DURATION
- Exposure duration: 4 hours (+/- S9 mix) and 18 hours (- S9 mix)
- 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: about 1.5


NUMBER OF CELLS EVALUATED: At least 100 well spread metaphases per culture were evaluated for cytogenetic damage on coded slides, except for the positive control in Experiment II without metabolic activation, where only 50 metaphases were evaluated.


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index and cell numbers


OTHER EXAMINATIONS:
- Determination of polyploidy: 500 per culture
- Determination of endoreplication: 500 per culture
Evaluation criteria:
Evaluation of the cultures was performed according to the OECD Guideline using NIKON microscopes with 100x 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. At least 100 well spread metaphases per culture were evaluated for cytogenetic damage on coded slides, except for the positive control in Experiment II without metabolic activation, where only 50 metaphases were evaluated.
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) and relative cell numbers were determined.

In addition, the number of polyploid cells in 500 metaphases per culture was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype). Additionally the number of endomitotic cells scored at the evaluation of polyploid cells was noticed and reported (% endomitotic metaphases).
Statistics:
Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05).
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
The test item, 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 presence of metabolic activation by S9 mix.
Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without S9 mix. In Experiment II the exposure period was 4 hours with S9 mix and 18 hours without S9 mix. The chromosomes were prepared 18 hours (Exp. I & II) after the start of treatment with the test item.
In each experimental group two parallel cultures were set up. At least 100 metaphases per culture were evaluated for structural chromosome aberrations, except for the positive control in Experiment II without metabolic activation, where only 50 metaphases were evaluated.
No relevant influence of the test item on pH value or osmolarity was observed.
Precipitation of the test item in culture medium was observed in Experiment I at preparation interval 18 hours with 96.0 µg/mL and above. In this experiment severe precipitation could be observed on the prepared slides beginning with 38.4 µg/mL in the absence and presence of S9 mix. In Experiment II after 18 hours continuous treatment precipitation occurred with 4.7 µg/mL and above without S9 mix and in the presence of S9 mix with 9.4 µg/mL and above. Severe precipitation on the slides was observed in the absence of S9 mix beginning with 18.8 µg/mL and 37.5 µg/mL in the presence of S9 mix. Evaluation of the mentioned slides was impossible due to interference of the cells by the precipitate.
In this study, neither reduced mitotic indices nor reduced cell numbers could be observed up to the highest evaluated concentrations of the test item, except in Experiment I, in the presence of S9 mix. In this part of the study, the cell numbers were slightly reduced after treatment with 6.1, 15.4 and 38.4 µg/mL (56.4, 72.8 and 76.5 % of control).
In Experiment I, in the absence and presence of S9 mix and in Experiment II in the absence 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 (1.0 - 3.5 % aberrant cells, excluding gaps) were close to the range of the solvent control values (2.0 - 3.5 % aberrant cells, excluding gaps) and within the range of the laboratory historical control data: 0.0 -4.0 % aberrant cells, excluding gaps. In Experiment II in the presence of S9 mix one single value slightly exceeded the range (6 % aberrant cells, excluding gaps) of the laboratory historical solvent control data (0.0 - 4.0 % aberrant cells, excluding gaps). However, this value was not statistically significant and no dose-dependency was observed with the evaluated concentrations. No relevant effect occurred in Experiment I and thus the finding is regarded as being without biological relevance.
In both experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (2.0 - 5.0 %) as compared to the rates of the solvent controls (2.7 - 3.2 %).
A slightly increased number of endomitotic cells was observed in both experiments (0.0 -1.5 %). As these effects were less frequent and not dose dependent they are regarded as biologically irrelevant.
In both experiments, either EMS (1000.0 or 600.0 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.

Summary of results of the chromosome aberration study with the test substance

Exp.

Preparation

Test item

Endomitotic

Polyploid

Cell numbers

Mitotic indices

Aberrant cells

 

interval

concentration

cells

cells

in %

in %

in %

 

 

in µg/mL

in %

in %

of control

of control

incl. gaps*

excl. gaps*

with exchanges

Exposure period 4 hrs without S9 mix

I

18 hrs

Solvent control1

0.1

3.2

100.0

100.0

3.5

3.5

1.0

 

 

Positive control2

n.d.

n.d.

n.d.

116.1

25.0

25.0S

17.5

 

 

2.5

0.0

5.0

108.5

101.6

1.0

1.0

0.5

 

 

6.1

0.0

3.7

123.2

92.5

2.5

2.0

0.0

 

 

15.4

0.0

3.1

100.8

112.9

2.5

2.0

1.5

Exposure period 18 hrs without S9 mix

II

18 hrs

Solvent control1

0.0

3.1

100.0

100.0

3.5

2.5

0.0

 

 

Positive control#3

n.d.

n.d.

n.d.

68.9

52.0

51.0S

15.0

 

 

1.2

0.0

2.9

88.0

112.4

1.5

1.0

0.0

 

 

2.4

0.0

2.8

94.9

111.6

4.0

3.5

0.5

 

 

4.7P

0.0

2.0

95.4

90.0

3.0

2.0

0.0

Exposure period 4 hrs with S9 mix

I

18 hrs

Solvent control1

0.1

3.1

100.0

100.0

2.0

2.0

1.5

 

 

Positive control4

n.d.

n.d.

n.d.

88.5

23.0

22.5S

9.5

 

 

6.1

1.1

3.5

56.4

121.8

2.5

2.5

1.0

 

 

15.4

1.0

2.7

72.8

101.7

1.5

1.5

0.5

 

 

38.4

0.3

2.3

76.5

108.1

2.0

1.0

1.0

II

18 hrs

Solvent control1

0.0

2.7

100

100

4.0

3.5

1.0

 

 

Positive control4

n.d.

n.d.

n.d.

48.6

16.5

15.0S

6.5

 

 

1.2

1.5

4.0

110.6

115.6

2.5

1.5

0.0

 

 

2.4##

0.2

2.6

111.9

103.8

6.5

6.0

1.3

 

 

4.7

0.1

2.9

103.7

93.4

3.5

3.0

1.0

 

 

9.4P

0.2

2.4

100.1

120.5

4.0

4.0

0.5

*     Inclusive cells carrying exchanges

#     Evaluation of 50 metaphases per culture

##    Evaluation of 200 metaphases per culture

n.d. Not determined

P     Precipitation occurred at the end of treatment

S     Aberration frequency statistically significant higher than corresponding control values

1     DMSO    0.5 % (v/v)

2         EMS 1000.0 µg/mL

3         EMS   600.0 µg/mL

4         CPA       1.4 µg/mL

Conclusions:
Interpretation of results (migrated information):
negative

In conclusion, the test substance is considered to be non-clastogenic in this chromosome aberration test in the absence and presence of metabolic activation, when tested up to precipitating or the highest evaluable concentrations.
Executive summary:

The test item, suspended in DMSO, was assessed for its potential to induce structural chromosome aberrations in V79cells of the Chinese hamster in vitro in two independent experiments. The following study design was performed:

 

Without S9 mix

With S9 mix

 

Exp. I

Exp. II

Exp. I

Exp. II

Exposure period

 4 hrs

18 hrs

 4 hrs

 4 hrs

Recovery

14 hrs

-

14 hrs

14 hrs

Preparation interval

18 hrs

18 hrs

18 hrs

18 hrs

In each experimental group two parallel cultures were set up. At least 100 metaphases per culture were evaluated for structural chromosome aberrations, except for the positive control in Experiment II without metabolic activation, where only 50 metaphases were evaluated.

The highest applied concentration (600.0 µg/mL; approx. 1.5 mM) was chosen with regard to the ability to formulate a homogeneous suspension of the test item in an appropriate solvent.

In the evaluable slides neither in the absence nor in the presence of S9 mix the mitotic index and/or cell numbers were reduced indicating no cytotoxicity.

In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item.

No relevant increase in polyploid metaphases was found after treatment with the test item as compared to the frequencies of the control cultures.

Appropriate mutagens were used as positive controls. They induced statistically significantincreases (p < 0.05) in cells with structural chromosome aberrations.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2020
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
See read across justification document in 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)
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: In vitro mammalian cell gene mutation test using the Hprt gene
Target gene:
HPRT
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
Chinese Hamster (Cricetulus griseus) ovary cell line CHO-K1,
(ATCC CCL-61, Lot 4765275)
Metabolic activation:
with and without
Untreated negative controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
Details on test system and experimental conditions:
Cells were grown in tissue culture flasks at 37 ± 1 °C in a humidified carbon dioxide incubator (5 ± 0.2 % CO2 in air)
Rationale for test conditions:
Test approaches currently accepted under the OECD for the assessment of mammalian cell gene mutation involve the use of Chinese Hamster Ovary (CHO) cell line. This cell line has been demonstrated to be sensitive to the mutagenic activity of a variety of chemicals.

Established CHO cell line is useful in in vitro gene mutation testing because it is easily cultured in standard medium, has a small number of large chromosomes each with a more or less distinctive morphology and a relatively short cycle time
Evaluation criteria:
CRITERIA FOR ACCEPTABILITY OF THE TEST
The assay will be considered valid if the following criteria are met:
a) The concurrent vehicle control data is within the range of the laboratory historical control data.
b) The concurrent positive control substances should induce responses that are compatible with those generated in the historical positive control data base and produce a statistically significant increase compared with the concurrent vehicle control.
c) Two experimental conditions are tested unless one results in positive response.
d) Adequate number of cells and analyzable concentrations are tested under each of the experimental conditions.
e) The criteria for the selection of top concentration are consistent with those described in the guideline.

EVALUATION AND INTERPRETATION OF RESULTS
When all the validity criteria are fulfilled:

1. A test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
• At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent vehicle control
• The increase is concentration-dependent when evaluated with an appropriate trend test
• Any of the results are outside the distribution of the historical vehicle control data

When all of these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.

2 A test chemical is considered to be clearly negative if, in all experimental conditions examined:

• None of the test concentrations exhibits a statistically significant increase compared with the concurrent vehicle control
• There is no concentration-related increase when evaluated with an appropriate trend test
• All results are inside the distribution of the historical vehicle control data

The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Conclusions:
It is concluded that the test item does not have the potential to induce gene mutation in CHO-K1 cells at the tested concentrations and under the conditions of testing employed.
Executive summary:

 

The genotoxic potential of the test item induce gene mutation in mammalian cells was evaluated using Chinese Hamster ovary (CHO) cells.

 

The study consisted of a preliminary cytotoxicity test and a definitive gene mutation test. The gene mutation test comprised of two independent experiments, one each in the presence and absence of metabolic activation system (S9 fraction prepared from Aroclor 1254 induced rat liver).

 

The test item was insoluble in sterile water and formed a free flowing suspension in Dimethyl sulfoxide (DMSO) at
200 mg/mL.

 

In a preliminary cytotoxicity test for the selection of test concentrations for the gene mutation assay, the Relative Survival was 27 and 32 % at the
1000 µg/mL, in the presence and absence of metabolic activation, respectively. There was precipitation of the test item in the test medium at and above
1000 µg/mL, both in the presence and absence of metabolic activation. There was no appreciable change in the pH and osmolality of test medium. Based on these observations a maximum of 1500 µg/mL was tested in the gene mutation assay.
 

 

In the gene mutation test, CHO-K1 cells were exposed to the test item in duplicate at concentrations of 23.44, 93.75, 375 and 1500 µg/mL of the medium for 3 hours in the presence (Experiment 1) and absence (Experiment 2) of metabolic activation. In a similar way, a concurrent vehicle control (DMSO) and a positive control, 3-methylcholanthrene (Experiment 1) were also tested in duplicate.

 

There was no evidence of induction of gene mutations in any of the test item treated cultures either in the presence or absence of metabolic activation. The positive control in experiment 1 produced a statistically significant increase in the frequencies of mutants, under identical conditions.

 

The results of the forward gene mutation test at the hprt-locus indicated that the test item was non-mutagenic under the conditions of this study

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2020
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
See read across justification document in chpter 13
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: In vitro mammalian cell gene mutation test using the Hprt gene
Target gene:
HPRT
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
Chinese Hamster (Cricetulus griseus) ovary cell line CHO-K1,
(ATCC CCL-61, Lot 4765275)
Metabolic activation:
with and without
Untreated negative controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
Details on test system and experimental conditions:
Cells were grown in tissue culture flasks at 37 ± 1 °C in a humidified carbon dioxide incubator (5 ± 0.2 % CO2 in air)
Rationale for test conditions:
Test approaches currently accepted under the OECD for the assessment of mammalian cell gene mutation involve the use of Chinese Hamster Ovary (CHO) cell line. This cell line has been demonstrated to be sensitive to the mutagenic activity of a variety of chemicals.

Established CHO cell line is useful in in vitro gene mutation testing because it is easily cultured in standard medium, has a small number of large chromosomes each with a more or less distinctive morphology and a relatively short cycle time
Evaluation criteria:
CRITERIA FOR ACCEPTABILITY OF THE TEST
The assay will be considered valid if the following criteria are met:
a) The concurrent vehicle control data is within the range of the laboratory historical control data.
b) The concurrent positive control substances should induce responses that are compatible with those generated in the historical positive control data base and produce a statistically significant increase compared with the concurrent vehicle control.
c) Two experimental conditions are tested unless one results in positive response.
d) Adequate number of cells and analyzable concentrations are tested under each of the experimental conditions.
e) The criteria for the selection of top concentration are consistent with those described in the guideline.

EVALUATION AND INTERPRETATION OF RESULTS
When all the validity criteria are fulfilled:

1. A test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
• At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent vehicle control
• The increase is concentration-dependent when evaluated with an appropriate trend test
• Any of the results are outside the distribution of the historical vehicle control data

When all of these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.

2 A test chemical is considered to be clearly negative if, in all experimental conditions examined:

• None of the test concentrations exhibits a statistically significant increase compared with the concurrent vehicle control
• There is no concentration-related increase when evaluated with an appropriate trend test
• All results are inside the distribution of the historical vehicle control data

The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Conclusions:
It is concluded that the test item does not have the potential to induce gene mutation in CHO-K1 cells at the tested concentrations and under the conditions of testing employed.
Executive summary:

 

The genotoxic potential of the test item induce gene mutation in mammalian cells was evaluated using Chinese Hamster ovary (CHO) cells.

 

The study consisted of a preliminary cytotoxicity test and a definitive gene mutation test. The gene mutation test comprised of two independent experiments, one each in the presence and absence of metabolic activation system (S9 fraction prepared from Aroclor 1254 induced rat liver).

 

The test item was insoluble in sterile water and formed a free flowing suspension in Dimethyl sulfoxide (DMSO) at
200 mg/mL.

 

In a preliminary cytotoxicity test for the selection of test concentrations for the gene mutation assay, the Relative Survival was 27 and 32 % at the
1000 µg/mL, in the presence and absence of metabolic activation, respectively. There was precipitation of the test item in the test medium at and above
1000 µg/mL, both in the presence and absence of metabolic activation. There was no appreciable change in the pH and osmolality of test medium. Based on these observations a maximum of 1500 µg/mL was tested in the gene mutation assay.
 

 

In the gene mutation test, CHO-K1 cells were exposed to the test item in duplicate at concentrations of 23.44, 93.75, 375 and 1500 µg/mL of the medium for 3 hours in the presence (Experiment 1) and absence (Experiment 2) of metabolic activation. In a similar way, a concurrent vehicle control (DMSO) and a positive control, 3-methylcholanthrene (Experiment 1) were also tested in duplicate.

 

There was no evidence of induction of gene mutations in any of the test item treated cultures either in the presence or absence of metabolic activation. The positive control in experiment 1 produced a statistically significant increase in the frequencies of mutants, under identical conditions.

 

The results of the forward gene mutation test at the hprt-locus indicated that the test item was non-mutagenic under the conditions of this study

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

No mutagenic or clastogenic effects were detected.