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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Mutagenicity in bacteria:

The test item showed no mutagenic activity in both experiments (plate incorporation assay, preincubation assay) each with and without metabolic activation.

Mutagenicity in mammalian cells:

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.

Chromosomal Aberrations in mammalian cells:

There was no evidence of statistically significant induction of chromosomal aberrations, excluding gaps, either in the presence or in the absence of metabolic activation. In each of the experiments, under identical conditions, the respective positive control substances produced a large and statistically significant increase in aberrant metaphases.

The study indicated that the test item was not clastogenic at the concentrations tested.

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:
From 2 MAR 2005 to 17 MAR 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: deionised water
- Justification for choice of solvent/vehicle: solubility properties of the solvent
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:
no cytotoxicity, but tested up to precipitating concentrations
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
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

1000- 5000

1000- 5000

1000- 5000

1000- 5000

TA 1537

1000- 5000

1000- 5000

1000- 5000

1000- 5000

TA 98

1000- 5000

1000- 5000

1000- 5000

1000- 5000

TA 100

1000- 5000

1000- 5000

1000- 5000

1000- 5000

TA 102

1000- 5000

1000- 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 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 cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2021
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
Arocalor
Test concentrations with justification for top dose:
Based on these observations and preliminary cytotoxicity test results, it was decided to test up to a maximum of 250 µg/mL in the presence and absence of metabolic activation with the 3-hour exposure and in the absence of metabolic activation with the 21-hours exposure period in the chromosomal aberration assay.
Vehicle / solvent:
Sterile water
Untreated negative controls:
yes
Positive controls:
yes
Details on test system and experimental conditions:
Chinese Hamster (Cricetulus griseus) ovary cell line CHO-K1, (ATCC CCL-61, Lot 63830396) with a doubling time approximately 12 -14 hours and modal chromosome number of 20 was used as the test system.

The cell line was tested for mycoplasma in the testing facility. The karyotype analysis of this cell line was periodically performed and documented.
Cells were grown in T- 25 cm2 and T-75 cm2 flasks at 37 ± 1 °C in a carbon dioxide incubator (5 ± 0.2 % CO2 in air)
Evaluation criteria:
Metaphases of all concentrations of the test item, the positive and vehicle control cultures were scored.
Statistics:
Statistical analysis of the experimental data was carried out using validated SYSTAT Statistical package ver.12.0. Data were analyzed for proportions of aberrant metaphases in each sample excluding gaps as aberrations. Pooled data from each test concentration and the positive control were compared with the vehicle control using Fischer exact test. All analysis and comparisons were evaluated at 5 % (p < 0.05) level.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Conclusions:
The test item, C.I. Pigment Yellow 175 was not clastogenic in CHO-K1 cells at the tested concentration and under the conditions of testing employed.
Executive summary:

The clastogenic potential of the test item, C.I. Pigment Yellow 175 to induce chromosomal aberrations in mammalian cells was evaluated using cultured Chinese Hamster Ovary (CHO-K1) cells in the presence and absence of an exogenous metabolic activation system (S9 fraction prepared from Aroclor 1254 induced rat liver).

 

The study consisted of a preliminary cytotoxicity test and a chromosome aberration assay. Chromosome aberration assay consisted of three independent experiments: Experiments 1 and 2 in the presence and absence of metabolic activation system with 3-hour exposure, respectively, and Experiment 3 in the absence of metabolic activation system with 21-hour exposure.

 

The test item formed a free flowing suspension in sterile water at 200 mg/mL and a pasty suspension in DMSO at 200 mg/mL. Hence, sterile water was selected as the vehicle of choice for the chromosomal aberration test.

 

In a preliminary cytotoxicity test for the selection of test concentrations for the chromosomal aberration assay, the test item did not exhibit the required level of cytotoxicity (reduction in the cell growth by 55± 5 % of the concurrent vehicle control)up to the highest tested concentration of 250µg/mL,both, in the presence and absence of metabolic activation (short term treatment) and in the absence of metabolic activation (long term treatment).

 

Based on these observations and preliminary cytotoxicity test results, it was decided to test up to a maximum of 250 µg/mL in thepresence and absence of metabolic activation with the 3-hour exposure and in the absence of metabolic activation with the 21-hours exposure period in the chromosomal aberration assay.

 

In the chromosomal aberration assay, CHO-K1 cells were exposed to the test item in duplicate at the concentrations of 15.63, 62.5 and 250mg/mL in Experiments 1, 2 and 3 of the chromosomal aberration assay. Concurrent vehicle (SW) and positive controls (cyclophosphamide monohydrate in the presence of metabolic activation and ethyl methanesulfonate in the absence of metabolic activation at 21 hour exposure) were also tested in duplicate. In each case, the cells in C-metaphase were harvested at approximately 21 hours after the start of the treatment and slides were prepared for chromosomal analysis.

 

At the highest concentrations tested, the reduction in cell growth as RICC was 47, 43 and 53 % in experiments 1, 2 and 3, respectively, compared to the vehicle control.

 

A total of 300 metaphases each from the SW control, each treatment level and the positive controls were evaluated for chromosomal aberrations.

 

There was no evidence of statistically significant induction of chromosomal aberrations, excluding gaps, either in the presence or in the absence of metabolic activation. In each of these experiments, under identical conditions, the respective positive control substances produced a large and statistically significant increase in aberrant metaphases.

The study indicated that the test item, C.I. Pigment Yellow 175 was not clastogenic at the concentrations tested and under the conditions of testing

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
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
Conclusions:
Interpretation of results (migrated information):
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 cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2021
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Please, see chapter 13: Read Across Justification
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
Arocalor
Test concentrations with justification for top dose:
Based on these observations and preliminary cytotoxicity test results, it was decided to test up to a maximum of 250 µg/mL in the presence and absence of metabolic activation with the 3-hour exposure and in the absence of metabolic activation with the 21-hours exposure period in the chromosomal aberration assay.
Vehicle / solvent:
Sterile water
Untreated negative controls:
yes
Positive controls:
yes
Details on test system and experimental conditions:
Chinese Hamster (Cricetulus griseus) ovary cell line CHO-K1, (ATCC CCL-61, Lot 63830396) with a doubling time approximately 12 -14 hours and modal chromosome number of 20 was used as the test system.

The cell line was tested for mycoplasma in the testing facility. The karyotype analysis of this cell line was periodically performed and documented.
Cells were grown in T- 25 cm2 and T-75 cm2 flasks at 37 ± 1 °C in a carbon dioxide incubator (5 ± 0.2 % CO2 in air)
Evaluation criteria:
Metaphases of all concentrations of the test item, the positive and vehicle control cultures were scored.
Statistics:
Statistical analysis of the experimental data was carried out using validated SYSTAT Statistical package ver.12.0. Data were analyzed for proportions of aberrant metaphases in each sample excluding gaps as aberrations. Pooled data from each test concentration and the positive control were compared with the vehicle control using Fischer exact test. All analysis and comparisons were evaluated at 5 % (p < 0.05) level.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Conclusions:
The test item, C.I. Pigment Yellow 175 was not clastogenic in CHO-K1 cells at the tested concentration and under the conditions of testing employed.
Executive summary:

The clastogenic potential of the test item, C.I. Pigment Yellow 175 to induce chromosomal aberrations in mammalian cells was evaluated using cultured Chinese Hamster Ovary (CHO-K1) cells in the presence and absence of an exogenous metabolic activation system (S9 fraction prepared from Aroclor 1254 induced rat liver).

 

The study consisted of a preliminary cytotoxicity test and a chromosome aberration assay. Chromosome aberration assay consisted of three independent experiments: Experiments 1 and 2 in the presence and absence of metabolic activation system with 3-hour exposure, respectively, and Experiment 3 in the absence of metabolic activation system with 21-hour exposure.

 

The test item formed a free flowing suspension in sterile water at 200 mg/mL and a pasty suspension in DMSO at 200 mg/mL. Hence, sterile water was selected as the vehicle of choice for the chromosomal aberration test.

 

In a preliminary cytotoxicity test for the selection of test concentrations for the chromosomal aberration assay, the test item did not exhibit the required level of cytotoxicity (reduction in the cell growth by 55± 5 % of the concurrent vehicle control)up to the highest tested concentration of 250µg/mL,both, in the presence and absence of metabolic activation (short term treatment) and in the absence of metabolic activation (long term treatment).

 

Based on these observations and preliminary cytotoxicity test results, it was decided to test up to a maximum of 250 µg/mL in thepresence and absence of metabolic activation with the 3-hour exposure and in the absence of metabolic activation with the 21-hours exposure period in the chromosomal aberration assay.

 

In the chromosomal aberration assay, CHO-K1 cells were exposed to the test item in duplicate at the concentrations of 15.63, 62.5 and 250mg/mL in Experiments 1, 2 and 3 of the chromosomal aberration assay. Concurrent vehicle (SW) and positive controls (cyclophosphamide monohydrate in the presence of metabolic activation and ethyl methanesulfonate in the absence of metabolic activation at 21 hour exposure) were also tested in duplicate. In each case, the cells in C-metaphase were harvested at approximately 21 hours after the start of the treatment and slides were prepared for chromosomal analysis.

 

At the highest concentrations tested, the reduction in cell growth as RICC was 47, 43 and 53 % in experiments 1, 2 and 3, respectively, compared to the vehicle control.

 

A total of 300 metaphases each from the SW control, each treatment level and the positive controls were evaluated for chromosomal aberrations.

 

There was no evidence of statistically significant induction of chromosomal aberrations, excluding gaps, either in the presence or in the absence of metabolic activation. In each of these experiments, under identical conditions, the respective positive control substances produced a large and statistically significant increase in aberrant metaphases.

The study indicated that the test item, C.I. Pigment Yellow 175 was not clastogenic at the concentrations tested and under the conditions of testing

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From 2 NOV 2004 to 27 DEC 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD TG 476)
Justification for type of information:
Please see Chapter 13: Read across Justification
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
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
Conclusions:
Interpretation of results (migrated information):
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 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

The test material did not cause mutagenic or clastogenic effects in bacterial or mammalian cells