Calcium 4-chloro-2-(5-hydroxy-3-methyl-1-(3-sulfonatophenyl)pyrazol-4-ylazo)-5-methylbenzenesulfonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item did not cause a significant increase in the number of revertant colonies with any of the tester strains in the Salmonella typhimurium reverse mutation assay (plate incorporation test) either in the absence or presence of metabolic activation. No dose dependent effect was observed. Therefore the test item is not considered to be mutagenic under the conditions in this Salmonella typhimurium reverse mutation assay.

The test item was considered not to induce structural chromosomal aberration but to induce numerical chromosomal aberration under the present test conditions.

In the gene mutation test, CHO-K1 cells were exposed to CI Pigment Yellow 191 in duplicate at concentrations of 15.63, 31.25, 62.5and 125 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 with the test item indicated that the test item was non-mutagenic under the conditions of this study

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Well performed GLP and OECD guideline study, but using a different set of tester strains

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Using a different set of tester stains

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:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254(R) induced rat liver S9-mix

Test concentrations with justification for top dose:

Experiment I: 0, 4, 20, 100, 500, 2500, 10000 µg/plate
Experiment II: 0, 4, 20, 100, 500, 2500, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Na-azide, 9-aminoacridine, 2-nitrofluorene, benzo[a]pyrene, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION:
in agar (plate incorporation)

DURATION
- Exposure duration: incubation for at least 48 to 72 hours at 37°C

NUMBER OF REPLICATIONS: 3 plates per strain and dose level, including controls, two independent experiments

Evaluation criteria:

Positive:

The test compound cause a significant increase in the number of revertant colonies with any of the tester strains either in the absence or presence
of S-9 Mix.

A dose dependent effect was obtained.


Negative:

The test compound does not cause a significant increase in the number of revertant colonies with any of the tester strains either in the absence or presence
of S-9 Mix.

No dose dependent effect was obtained.

Statistics:

Arithmetic means of the counted colonies were calculated.

Key result

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

incipient at 500 µg/plate without metabolic acivation

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

The test item did not cause a significant increase in the number of revertant colonies with any of the tester strains in the Salmonella typhimurium reverse mutation assay (plate incorporation test) either in the absence or presence of metabolic activation. No dose dependent effect was observed. Therefore the test item is not considered to be mutagenic under the conditions in this Salmonella typhimurium reverse mutation assay.

Executive summary:

Mutagenic activity of the test item was investigated in Salmonella typhymurium strains 1535, 1537, 98, 100 and 1538 with and without metabolic activation (induced rat liver S9-mix) at concentrations of 0, 4, 20, 100, 500, 2500, 5000 and 10 000 µg/plate (experiment I) and concentrations of

0, 4, 20, 100, 500, 2500, 5000 µg/plate (experiment II). Each concentration was tested in triplicate. Initial Toxic effects and precipitation were evident at 500 µg/plate.

Under the conditions tested the test compound did not cause a significant increase in the number of revertant colonies and no dose dependent effect was observed. Therefore the test item is not considered mutagenic under the conditions in this Salmonella typhimurium reverse mutation assay.

Reference 2

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

well performed GLP and OECD-like guideline study

Qualifier:

according to guideline

Guideline:

other: The Notification on Partial Revision of Testing Methods Relating to New Chemical Substances" (Notification No.700 of Kanpogyo, No.1039 of Yakuhatsu, and No. 1014 of 61 Kikyoku, December 5, 1986)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

other: Chinese hamster lung fibroblasts (CHL cells. clone No.11)

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and 5,6-benzoflavone induced rat liver S9

Test concentrations with justification for top dose:

75, 150, and 300 µg/ml for the 24h treatment, and 47.5, 95, and 190 µg/ml for the 48h treatment by the direct method without metabolic activation, and 750, 1500, and 3000 µg/ml for the metabolic activation method.

Vehicle / solvent:

Eagle's minimum essential medium supplemented with newborn calf serum at a rate of 10% (v/v). (10% NCS/MEM)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

with and without metabolic activation , respectively

Details on test system and experimental conditions:

Dose levels:
Test substance: The highest concentration decided in the dose determination test was employed as the maximum dose for the chromosomal aberration test. The test was carried out at three dose levels.
Positive control substances: Direct method MMC 0.05 µg/ml, Metabolic activation method CPA 10 µg/ml

Direct method:
After removal of the medium from two- or three-day-old cultures, 5 ml of the medium used to dissolve the test substance was added and incubated for 24 or 48 h. Two hours prior to the end of incubation, colcemid was added to the medium to give a concentration of 0.1 µg/ml. Then chromosome preparations were made.
A non-treated group and a MMC-treated group were used as a negative control and as positive control, respectively.

Metabolic activation method:
After removal of the medium from three-day-old cultures, 3 ml of S9 Mix/MEM which dissolved the test substance was added and treated for 6 h. After S9 Mix/MEM had been removed. the dishes were rinsed three times with 2 ml of PBS(-), and 5 ml of 10% NCS/MEM was added. The cells were cultured for 18 h. Two hours prior to the end of incubation, colcemid was added to give a concentration of 0.1 µg/ml. Then chromosome preparations were made.
A non-treated group and a CPA-treated group were used as a negative control and as positive control, respectively.
As the control for this test, a culture without S9 Mix was treated with the test substance at the same dose levels and treatment times to clarify the effect of metabolic activation.


MMC: mitomycin C
PBS (-): Ca(2+), Mg(2+)-free phosphate buffered saline
CPA: cyclophosphamide
NCS/MEM: Eagle's minimum essential medium, supplemented with newborn calf serum

Evaluation criteria:

The incidence of cells with aberrations including gap was:

less than 5 %: negative (-)
5% or more, less than 10%: suspect positive ( ±)
10% or more, less than 20%: positive (+)
20% or more, less than 50%: positive (++)
50% or more: positive (+++)

The test substance was judged to be positive for induction of chromosomal aberration when the incidence of 10% or more was doserelated or reproducible. The test was reexamined whenever the result was suspect positive, positive at only one dose level, or not evidently dose-related.

Species / strain:

other: Chinese hamster lung fibroblasts (CHL cells. clone No.11)

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

(structural aberration)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Chromosomal aberration test by the direct method

24-hour treatment

The incidences of cells with structural chromosomal aberrations including gap were 1.0% at 75µg/ml, 1.5% at 150µg/ml and 0.5% at 300µg/ml, suggesting a negative effect. The incidence in the non-treated group was 0.0%, i.e. within the normal range. The positive control group treated with MMC showed an evident incidence of structural aberration of 44.0%.

The incidence of polyploid cells was negative in each treatment group.

 

48-hour treatment

The incidences of cells with structural chromosomal aberrations including gap were 1.5% at 47.5µg/ml, 1.5% at 95µg/ml and 0.0% at 190µg/ml, suggesting a negative effect. The incidence in the non-treated group was 0.5%, i.e. within the normal range. The positive control group treated with MMC showed an evident incidence of structural aberration of 49.5%.

The incidence of polyploid cells was negative in each treatment group.

 

Chromosomal aberration test by the metabolic activation method

With S9 Mix

The incidences of cells with structural aberrations including gap were 1.0% at 750µg/ml, 0.0% at 1500µg/ml and 2.0% at 3000µg/ml. suggesting a negative effect. The incidence in the non-treated group was 0.0%, i.e. within the normal range. The positive control group treated with CPA showed an evident incidence of chromosomal aberration of 23.0%.

The incidence of polyploid cells was negative in each treatment group.

 

Without S9 Mix

In the medium without S9 Mix and with the same concentration and treatment time as S9 Mix added group, the incidences of structural chromosomal aberrations including gap were 0.0% at 750µg/ml and 0.0% at 1500µg/ml, suggesting a negative effect. Mitotic metaphases were not observed at dose of 3.000µg/ml, because cytotoxicity of the test substance was strong. The incidences of polyploid cells were 20.5% at 750µg/ml, suggesting a positive effect, and 9.5% at 1.500µg/ml, suggesting a suspect positive effect. In the non-treated group and positive control group, the incidences were 1.0 and 1.5 %, i.e. within the normal range, respectively.

 

Reexamination test without S9 Mix

The incidences of polyploid cells were 10.5% at 750µg/ml of test substance, suggesting a positive effect, and 6.5% at 500µg/ml and 7.0% at 1000µg/ml, suggesting suspect positive effect. In the non-treated group and positive control group, the incidences were 0.0 and 0.0%, i.e. within the normal range, respectively. The incidence of cells with structural aberrations was negative in each treatment group. The positive control group treated with S9 Mix showed an evident incidence of structural chromosomal aberration of 40.0%.

Conclusions:

Interpretation of results (migrated information):
negative structural chromosomal aberration
positive numerical chromosomal aberration in the absence of S9 Mix

Based on the findings PY-HGR was considered not to induce structural chromosomal aberration but to induce numerical chromosomal aberration under the present test conditions.

Executive summary:

The chromosomal aberration effect of PY-HGR was investigated using Chinese hamster lung fibroblasts (CHL cells), employing mitomycin C (MMC) and Cyclophosphamide (CPA) as the positive controls for the tests without and with metabolic activation, respectively.

Inhibition tests on cell growth and cell division were carried out to determine the dose levels of the test substance. From the results of these tests, chromosomal aberration tests were carried out using 75, 150, and 300µg/ml of the test substance for 24-h treatment, and 47.5, 95 and 190µg/ml for 48-h treatment by the direct method (without metabolic activation), and 750, 1500 and 3000µg/ml for the metabolic activation method. As the positive control, MMC was employed at 0.05µg/ml for 24-h treatment and 48-h treatment by the direct method, and CPA was used at 10µg/ml by the metabolic activation method.

It was shown that the test substance possessed a chromosomal aberration-negative effect at all concentrations in 24-h and 48-h treatments by the direct method and in the treatment with S9 Mix by the metabolic activation method, but possessed a numerical aberration positive effect at 750 µg/ml and suspect positive effect at 1500µg/ml in the treatment without S9 Mix by the metabolic activation method.

Therefore reexamination test of chromosomal aberration was carried out to confirm a numerical aberration positive effect using 500, 750, and 1000µg/ml of the test substance for the treatment without S9 Mix by the metabolic activation method.

Test substance showed a numerical aberration positive effect in the reexamination test within the dose range of 500-750µg/ml of the test substance, and incidence of cells with numerical aberration increased dose-dependently. The positive controls treated with MMC and CPA showed evident chromosomal aberrations.

From the results described above PY-HGR was considered not to induce structural chromosomal aberration but to induce numerical chromosomal aberration under the present test conditions.

Reference 3

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

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

incipient at 500 µg/plate without metabolic acivation

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Reference 4

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

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

other: Chinese hamster lung fibroblasts (CHL cells. clone No.11)

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

(structural aberration)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

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 63830396)

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Rat liver homogenate

Test concentrations with justification for top dose:

Based on the observations of the preliminary cytotoxicity test, following concentrations of the test item are selected for testing in the gene mutation assay:

Experiments 1 & 2 (3-hour Exposure in the Presence & Absence of Metabolic Activation, respectively)

a) 15.63 b) 31.25 c) 62.5 and d) 125 µg/mL (approximately factor of 2)

Vehicle / solvent:

DMSO

Untreated negative controls:

other: DMSO

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Details on test system and experimental conditions:

Chinese Hamster (Cricetulus griseus) ovary cell line CHO-K1,
(ATCC CCL-61, Lot 63830396) with a modal chromosome number 20 and a population doubling time of 12 to 14 hours was used.

This cell line is capable of developing resistance to 6-thioguanine (6TG) resulting from lack of hypoxanthine guanine phosphoribosyl transferase (hprt) enzyme activity as a result of mutation at the X chromosomes.

The cell line was tested for mycoplasma in the test facility. The karyotype analysis of this cell line is periodically performed and documented.

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.

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

Remarks on result:

other:

Conclusions:

It is concluded that the test item, C.I. Pigment Yellow 191 - nanoform 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 191 - nanoform to 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 formed a pasty lump in sterile water and 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 49 and 55 % at the 125 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 125 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 125 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 15.63, 31.25, 62.5and 125 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 the test item indicated that the test item wnon-mutagenic under the conditions of this study

Reference 6

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:

2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

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 63830396)

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Rat liver homogenate

Test concentrations with justification for top dose:

Based on the observations of the preliminary cytotoxicity test, following concentrations of the test item are selected for testing in the gene mutation assay:

Experiments 1 & 2 (3-hour Exposure in the Presence & Absence of Metabolic Activation, respectively)

a) 15.63 b) 31.25 c) 62.5 and d) 125 µg/mL (approximately factor of 2)

Vehicle / solvent:

DMSO

Untreated negative controls:

other: DMSO

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Details on test system and experimental conditions:

Chinese Hamster (Cricetulus griseus) ovary cell line CHO-K1,
(ATCC CCL-61, Lot 63830396) with a modal chromosome number 20 and a population doubling time of 12 to 14 hours was used.

This cell line is capable of developing resistance to 6-thioguanine (6TG) resulting from lack of hypoxanthine guanine phosphoribosyl transferase (hprt) enzyme activity as a result of mutation at the X chromosomes.

The cell line was tested for mycoplasma in the test facility. The karyotype analysis of this cell line is periodically performed and documented.

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.

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

Remarks on result:

other:

Conclusions:

It is concluded that the test item, C.I. Pigment Yellow 191 - nanoform 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 191 - nanoform to 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 formed a pasty lump in sterile water and 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 49 and 55 % at the 125 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 125 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 125 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 15.63, 31.25, 62.5and 125 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 the test item indicated that the test item wnon-mutagenic under the conditions of this study

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Oral administration of the test item did not lead to a substantial increase of micronucleated polychromatic erythocytes. Therefore, the test item was not mutagenic in the in vivo mouse micronucleus test

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Well performed GLP and OECD guideine study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain specifics: Hoe: NMRKf (SPF71)
- Source: Hoechst AG breeding colony
- Age at study initiation: 7 weeks
- Weight at study initiation: males: 29-36 g, mean 32.1 g, females: 21-29 g, mean 24.7 g
- Housing: in groups of five in Macrolon type 3 cages in fully air-conditioned room, softwood granulate
- Diet (e.g. ad libitum): rat/mouse standard diet Altromin 1324, ad libitum
- Water (e.g. ad libitum): tap water in plastic bottles, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): 55 ± 10 %
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: sesame oil (Oleum Sesami Ph. Eur. III, Pharm. Fabrik GmbH, Frankfurt/Main, Germany)
- Concentration of test material in vehicle: 10 % (w/v)
- Amount of vehicle (if gavage or dermal): 20 mL/kg body weight

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

the test substance suspensions were prepared fresh each day. 5000 mg were weight in a beaker, mixed with sesame oil, washed out in a 50 mL flask and topped up to the calibration mark.

Duration of treatment / exposure:

oral administration of two equal parts within 2 hours for 24 h and 48 h dosed group; one single oral administration for the 72 h dosed group.

Frequency of treatment:

See above

Post exposure period:

24 hours (dosed group, negative control and positive control), 48 hours (dosed group and negative control) and 72 hours (dosed group and negative control)

Remarks:

Doses / Concentrations:
2000
Basis:
nominal conc.
mg/kg body weight

No. of animals per sex per dose:

5 males and 5 females (10 animals) per dose group

Based on the results of a preliminary range finding study, a dose of 2000 mg/kg bw was the maximum applicable dose level.

Control animals:

yes, concurrent vehicle

Positive control(s):

For the cyclophosphamide stock solution 5 mL distilled water were added to 100 mg cyclophosphamide in an injection phial and shaken to form a clear solution. For administration 2 mL of the 2 % stock solution were mixed with 6 mL distilled water.
50 mg/kg body weight cyclophosphamide (Endoxan (R), 5 males and 5 females)

Tissues and cell types examined:

1000 polychromatic erythrocytes from the femoral bone marrow were counted for each animal.

Details of tissue and slide preparation:

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Animals were killed 24, 48 or 72 h after application

DETAILS OF SLIDE PREPARATION:
Removal of femoral bones and bone marrow from the proximal ends flushed into centrifuge tube containing about 3 ml foetal bovine serum, centrifugation (5 min at 1200 rpm), one drop of thoroughly mixed sediment smeared on a cleaned slide, air-dried for approx. 24 h, staining (methanol, May-Grünwalds solution, Giemsa) and coating with Entellan

METHOD OF ANALYSIS:
Number of cells with micronuclei and ratio of polychromatic to normochromatic erythrocytes
Statistical evaluation (see below)

Evaluation criteria:

The results of the treatment groups at each killing time were compared with corresponding control values.
95 % level of significance for comparisons.
Actual data were also compared with historical controls.

Statistics:

Wilcoxon (paired, two-sided) for the ratio of polychromatic to normochromatic erythrocytes.
Wilcoxon (paired, on-sided, increase) for the proportion of polychromatic and normochromatic erythrocytes with micronuclei

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

all animals free of clinical signs of toxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The dissection of the animals revealed yellow coloured content in stomach and intestinum.

RESULTS OF RANGE-FINDING STUDY

- Dose range: 2000 mg/kg body weight
- Other: lethality: 0 out of 3 males and 0 out of 3 females

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no statistically significant increase of micronucleated polychromatic erythrocytes in dosed animals
- Ratio of PCE/NCE (for Micronucleus assay): the ratio remained essentially unaffected by the test substance
- Statistical evaluation: see above

Mean mutation indices in polychromatic erythrocytes:

sex	sampling after dosing	dose [mg/kg bw]	mean mutation index
male	24 h	control	1.0
	24 h	2000	1.5
	24 h	positive control	29.0
female	24 h	control	1.0
	24 h	2000	1.8
	24 h	positive control	22.7
male	48 h	control	1.0
	48 h	2000	1.7
female	48 h	control	1.0
	48 h	2000	1.3
male	72 h	control	1.0
	72 h	2000	0.7
female	72 h	control	1.0
	72 h	2000	1.1

Conclusions:

Interpretation of results (migrated information): negative
Oral administration of the test item did not lead to a substantial increase of micronucleated polychromatic erythocytes. Therefore, the test item was not mutagenic in the in vivo mouse micronucleus test

Executive summary:

The test item was tested in the in vivo micronucleus test according to OECD 474. The test substance was suspended in sesame oil and dosed orally at 2000 mg per kg bodyweight to male and female mice, based on the results of the previously conducted dose range finding assay. According to the test procedure the animals were killed 24, 48 or 72 hours after administration.

Endoxan® was used as positive control substance and was administered orally at a dose of 50 mg per kg bodyweight.

 

The number of polychromatic and normochromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic/normochromatic erythrocytes in both male and female animals remained unaffected by the treatment with the test item and was statistically not different from the control values.

Endoxan® induced in both males and females a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity of the system.

The results indicate that, under the conditions of the present study, the test item is not mutagenic in the micronucleus test.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reason / purpose for cross-reference:

read-across source

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION:
Removal of femoral bones and bone marrow from the proximal ends flushed into centrifuge tube containing about 3 ml foetal bovine serum, centrifugation (5 min at 1200 rpm), one drop of thoroughly mixed sediment smeared on a cleaned slide, air-dried for approx. 24 h, staining (methanol, May-Grünwalds solution, Giemsa) and coating with Entellan

METHOD OF ANALYSIS:
Number of cells with micronuclei and ratio of polychromatic to normochromatic erythrocytes
Statistical evaluation (see below)

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

all animals free of clinical signs of toxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Pigment Yellow 191 did not reveal mutagenic effect in the bacterial reverse mutation assay and did not cause structural chromosomal aberrations in the in vitro mammalian chromosome aberration test. The numerical aberrations observed in the in vitro mammalian chromosome aberration test could not be verified in an higher-tier in vivo micronucleus test from which negative results were obtained. The test item was not mutagenic in mammalian cells in an HPRT assay.

Therefore Pigment Yellow 191 is considered not to be mutagenic and does not have to be classified for mutagenicity according to the criteria laid down in Directive 67/548/EEC and in Regulation (EC) No 1272/2008.