[1,3-dihydro-5,6-bis[[(2-hydroxy-1-naphthyl)methylene]amino]-2H-benzimidazol-2-onato(2-)-N5,N6,O5,O6]nickel

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three Bacerial Reverse Mutation Assayswere performed to investigate the potential of C.I. Pigment Orange 68 to induce gene mutations using the following tester strains:

key_471_1983_Hatano_58-I138 (M908):

Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and 1538, and the Escherichia coli strain WP2 uvrA, with and without metabolic activation.

key_471 E. coli_1982_RCC_012892:

Escherichia coli strain WP2 uvrA, with and without metabolic activation.

key_471 S. typhimurium_1982_RCC_012892:

TA 1535, TA 1537, TA 98, TA 100, and 1538,with and without metabolic activation.

Although each of these tests is incomplete (regarding tester strains or confirmation of negative results) the set in its entirety fulfills all reuirements of a fully valid study thus allowing the conclusion that the test item exerts no gene mutagenic activity in this test system.

 

Justification for selection of genetic toxicity endpoint
No single study was selected because in view of minor shortcomings in all available studies preference is given to the entirety of all available studies in order to get a fully valid data set.

Short description of key information:
AMES; key_471_1983_Hatano_58-I138 (M908): negative
AMES, key_471 E. coli_1982_RCC_012892: negative
AMES, key_471 S. typhimurium_1982_RCC_012892: negative

Endpoint Conclusion: No adverse effect observed (negative)

C.I. Pigment Orange 68 was not genotoxic in the mammalian cell gene mutation test (HPRT) in Chinese Hamster ovary cells (CHO-K1) when tested with and without rat liver S9 metabolic activation at concentrations up to 1000 µg/mL.

 

C.I. Pigment Orange 68 was not clastogenic in the mammalian cell chromosome aberration test in Chinese Hamster ovary cell (CHO-K1) when tested with and without rat liver S9 metabolic activation at concentrations up to 1000 µg/mL.

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:

other: well performed guideline like study with GLP but without confirmation of negative results by furher testing

Qualifier:

according to guideline

Guideline:

other: Green, 1976

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

AROCLOR 1254 induced rat liver S-9 mix

Test concentrations with justification for top dose:

1.58 / 5 / 15.8 / 50 / 158 / 500 / 1580 AND 5000 µg/plate

Vehicle / solvent:

DIMETHYLSULFOXIDE

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: METHYL METHANE SULFONATE, N-ETHYL-N-NITRO-N-NITROSOGUANIDINE, 2-AMINOANTHRACENE

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation

NUMBER OF REPLICATIONS: TRIPLICATE PLATES, without confirmation of negative results by further testing

Evaluation criteria:

SPECIAL ATTENTION WAS GIVEN TO A REPRODUCIBLE DOSE-EFFECT RELATIONSHIP.
A MUTAGENIC ACTIVITY IS ASSUMED IF AT LEAST A TWO FOLD INCREASE OF THE NUMBER OF INDUCED REVERTANTS IS OBTAINED IN COMPARISON WITH THE SPONTANEOUS REVERTANTS OF THE CORRESPONDING CONTROL.

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

IN CONCLUSION IT CAN BE STATED THAT DURING THIS IN VITRO ESCHERICHIA COLI REVERSE MUTATION ASSAY NO GENE MUTAGENIC ACTIVITY COULD BE DEMONSTRATED UNDER THE EXPERIMENTAL CONDITIONS REPORTED.

Executive summary:

THE TEST ITEM WAS TESTED FOR DETECTING ITS POTENTIAL GENE MUTAGENIC ACTIYITY ACCORDING TO THE PLATE INCORPORATION METHOD OF GREEN ET AL, USING THE ESCHERICHIA COLI STRAIN WP2UVRA.

THE TEST WAS PERFORMED WITH AND WITHOUT LIVER MICROSOMAL ACTIYATION. THE TEST MATERIAL WAS TESTED AT EIGHT CONCENTRATIONS: 1.58 / 5 / 15.8 / 50 / 158 / 500 / 1580 AND 5000 MICROGRAMS (MCG) PER PLATE. EACH COMPOUND CONCENTRATION INCLUDING CONTROLS WAS TESTED IN TRIPLICATE.

A VERY SLIGHT PRECIPITATION OF THE TEST MATERIAL WAS OBSERYED AFTER POURING THE CONTENT OF THE TEST TUBE OVER THE SURFACE OF THE SELECTIYE AGAR PLATE AT THE CONCENTRATIONS OF 1580 AND 5000 MCG PER PLATE. THE REVERTANTS WERE EASILY RECOGNIZABLE AND THE PLATES COULD BE EYALUATED.

UP TO THE HIGHEST TESTED DOSE, WITH AND WITHOUT LIVER MICROSOMAL ACTIYATION, NO RELEVANT INCREASE OF THE REVERTANT COLONY NUMBERS WAS OBTAINED IN COMPARISON WITH THE CORRESPONDING CONTROLS.

NO TOXIC EFFECTS OF THE TEST MATERIAL WERE OBSERVED.

Reference 2

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:

other: well performed guideline like study with GLP but without confirmation of negative results by furher testing

Qualifier:

according to guideline

Guideline:

other: Ames, 1975

Deviations:

not specified

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 induced rat liver S-9 mix

Test concentrations with justification for top dose:

1.58 / 5 / 15.8 / 50 / 158 / 500 / 1580 AND 5000 µg/plate

Vehicle / solvent:

DIMETHYLSULFOXIDE

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: METHYL METHANESULFONATE, 9-AMINOACRIDINE, 2-NITROFLUORENE, N-ETHYL-N-NITRO-N-NITROSOGUANIDINE, 2-AMINOANTHRACENE, BENZO-(A)-PYRENE

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation

NUMBER OF REPLICATIONS: TRIPLICATE PLATES, without confirmation of negative results by further testing

Evaluation criteria:

SPECIAL ATTENTION WAS GIVEN TO A REPRODUCIBLE DOSE-EFFECT RELATIONSHIP.

A MUTAGENIC ACTIVITY WAS ASSUMED IF AT LEAST A TWO FOLD (FOR TA 100: ONE AND HALF FOLD) INCREASE OF THE NUMBER OF INDUCED REVERTANTS WAS OBTAINED IN COMPARISON WITH THE SPONTANEOUS REVERTANTS OF THE CORRESPONDING CONTROLS.

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

IN CONCLUSION, IT CAN BE STATED THAT DURING THIS IN VITRO SALMONELLA/MAMMALIAN-MICROSOME ASSAY NO GENE MUTAGENIC ACTIVITY COULD BE DEMONSTRATED UNDER THE EXPERIMENTAL CONDITIONS REPORTED.

Executive summary:

THE TEST ITEM WAS TESTED FOR DETECTING ITS POTENTIAL GENE MUTAGENIC ACTIVITY ACCORDING TO THE PLATE INCORPORATION METHOD OF AMES ET AL. USING THE SALMONELLA THYPHIMURIUM STRAINS TA 98, TA 100, TA 1535, TA 1537 AND TA 1538.

THE TEST WAS PERFORMED WITH AND WITHOUT LIVER MICROSOMAL ACTIVATION. THE TEST MATERIAL WAS TESTED AT EIGHT CONCENTRATION: 1.58 / 5 / 15.8 / 50 / 158 / 500 / 1580 AND 5000 MICROGRAMS (MCG) PER PLATE. EACH COMPOUND CONCENTRATION INCLUDING CONTROLS WAS TESTED IN TRIPLICATE.

A VERY SLIGHT PRECIPITATION OF THE TEST MATERIAL WAS OBSERVED AFTER POURING THE CONTENT OF THE TEST TUBE OVER THE SURFACE OF THE SELECTIVE AGAR PLATE AT THE CONCENTRATIONS OF 1580 AND 5000 MCG PER PLATE. THE REVERTANTS WERE EASILY RECOGNIZABLE AND THE PLATES COULD BE EVALUATED.

UP TO THE HIGHEST TESTED DOSE, WITH AND WITHOUT MICROSOMAL ACTIVATION, NO RELEVANT INCREASE OF THE REVERTANT COLONY NUMBERS WAS OBTAINED IN ANY SALMONELLA THYPHIMURIUM STRAIN IN COMPARISON WITH THE CORRESPONDING CONTROLS.

NO TOXIC EFFECTS OF THE TEST MATERIAL WERE OBSERVED.

Reference 3

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:

other: well performed guideline study with GLP like affirmation but without confirmation of negative results by further testing

Qualifier:

according to guideline

Guideline:

other: Japan Industrial Safety and Health Law (ISHL)

Deviations:

not specified

GLP compliance:

not specified

Remarks:

GLP like statement which confirms, that the report provides a correct and faithful record of the results obtained.

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

PCB 500 induced rat liver S-9 mix

Test concentrations with justification for top dose:

10; 50; 100; 500; 1000; and 5000 µg/plate

Vehicle / solvent:

Dimethylsulfoxide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide, 2-Aminoanthracene, 9-Aminoacridine, 4-Nitro-o-phenylenediamine, Sodium azide

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation
NUMBER OF REPLICATIONS: duplicate plates, without confirmation of negative results by further testing

Evaluation criteria:

Significant differences in the number of the colonies between the test compound dosages and the control with any of the test strains used.

Statistics:

no data

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

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:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test with and without PCB 500 induced rat liver S9 mix using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and 1538, and the Escherichia coli strain WP2 uvrA.

Each concentration, including the controls, was tested in duplicate. The test item was tested at the following concentrations:

10; 50; 100; 500; 1000; and 5000 µg/plate

Significant differences were not observed in the number of the colonies between the test compound dosages and the control with any of the test strains used.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

 

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

according to OECD and GLP guidelines

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: In vitro Mammalian Chromosome Aberration

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: American Type Culture Collection, P. O. Box 1549, Manassas, VA 20108, USA

- Suitability of cells: Test approaches currently accepted under the OECD guidance for the assessment of mammalian cell chromosome damage involve the use of Chinese Hamster Ovary (CHO) cell line. This cell line has been demonstrated to be sensitive to the chromosome damage by a variety of chemicals.
An established CHO cell line is useful in in vitro cytogenetics 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.

- Cell cycle length, doubling time or proliferation index: 12 -14 hours
- Number of passages if applicable: within 10 passages
- Methods for maintenance in cell culture if applicable: Routine sub culture
- Modal number of chromosomes: 20

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Ham’s F-12 medium supplemented with sodium bicarbonate, antibiotics (penicillin, streptomycin and amphotericin), L-glutamine and 5 or 10% fetal bovine serum (F-12 FBS 5/10).
- Properly maintained: [yes]
- Periodically checked for Mycoplasma contamination: [yes]
- Periodically checked for karyotype stability: [yes]

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9 homogenate was used as the metabolic activation system.

Test concentrations with justification for top dose:

Based on the observations of the preliminary cytotoxicity test, following concentrations of the test item were selected for testing in the chromosomal aberration assay:
a) 250 b) 500 and c) 1000 µg/mL (factor of 2)

Vehicle / solvent:

DMSO- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was insoluble in sterile water and formed a free flowing suspension in DMSO at 100 mg/mL.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension
- Cell density at seeding (if applicable): 10 lakhs

DURATION
- Exposure duration: 3 hours

SPINDLE INHIBITOR (cytogenetic assays): Colchicine

NUMBER OF REPLICATIONS: Two

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
The cell suspension was dropped onto a clean chilled slide, flame dried and dried on a slide warmer maintained at approximately 35 to 40 °C. The slides were marked with the study number, treatment group, activation, experiment number, replicate number and the slide number with a diamond point marker. Five slides were prepared per replicate.

The slides were stained with freshly prepared Giemsa stain in water, for 120 minutes, washed in water, air dried, immersed in xylene and mounted with DPX. The slides were then coded by an individual not involved in scoring process before evaluation.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 300 per group

DETERMINATION OF CYTOTOXICITY
- Method: othrer: Cell count

OTHER EXAMINATIONS:
- Determination of polyploidy: Cell containing more than the diploid number (2n) of chromosomes, in exact multiples of the haploid number (n) eg., triploid = 3n, tetraploid = 4n, etc
- Determination of endoreplication: Chromatid alignment is maintained in a cell in which the chromosomes have duplicated but the cell has failed to cleave, a form of polyploidy.

Rationale for test conditions:

The purpose of the in vitro mammalian chromosome aberration test was to evaluate the potential of the test item to induce structural chromosome aberrations in cultured mammalian cells.

Evaluation criteria:

The assay is considered valid if the following criteria are met:

• The concurrent negative control is considered acceptable for addition to the laboratory historical negative control database.
• Concurrent positive controls 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 negative control.
• Cell proliferation criteria in the solvent control should be fulfilled as per OECD TG 473.
• All three experimental conditions were tested unless one resulted in positive results.
• Adequate number of cells and concentrations are analysable.
• The criteria for the selection of top concentration are consistent with those described in OECD TG 473.

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Summary Results of Chromosomal Aberration Assay - Experiment 1

Refer Appendices 3 and 4

Treatment (µg/mL)	No. of metaphases scored	No. (%) of metaphases with aberrations^@						Total No.(%) of aberrant metaphases^*		Cell Growth Inhibition (%)
		Gaps		Breaks		Exchanges		Including Gaps	Excluding Gaps
		Cs	Ct	Cs	Ct	Cs	Ct
DMSO	300	0	1 (0.3)	1 (0.3)	0	0	0	2 (0.7)	1 (0.3)	0
250	300	1 (0.3)	2 (0.7)	0	0	0	0	3 (1.0)	0	22
500	300	2 (0.7)	2 (0.7)	0	1 (0.3)	0	0	3 (1.0)	1 (0.3	28
1000	300	1 (0.3)	1 (0.3)	1 (0.3)	1 (0.3)	0	0	3 (1.0)	1 (0.3)	32
CPA 55	300	18 (6.0)	17 (5.7)	54 (18.0)	101 (33.7)	38 (12.7)	25 (8.3)	166  (55.3)	+ 166  (55.3)	45

*: Metaphase plate with one or more than one aberrations considered as one metaphase plate with aberrations

@: values are the sum of two replicates and values in parenthesis represent %  

Cs: Chromosome type; Ct: Chromatid type; CPA: Cyclophosphamide monohydrate; +: Significantly higher than control (p <0.05) by Fischer exact test

Note: There were no incidences of polyploidy and endoreduplicated cells

SummaryResults of Chromosomal Aberration Assay - Experiment 2

Refer Appendices 3 and 5

Treatment (µg/mL)	No. of metaphases scored	No. (%) of metaphases with aberrations^@						Total No. (%) of aberrant metaphases^*		Cell Growth Inhibition (%)
		Gaps		Breaks		Exchanges		Including Gaps	Excluding Gaps
		Cs	Ct	Cs	Ct	Cs	Ct
DMSO	300	0	0	0	0	0	0	0	0	0
250	300	0	0	0	0	0	0	0	0	20
500	300	1 (0.3)	0	0	0	0	0	1 (0.3)	0	29
1000	300	0	1 (0.3)	0	0	0	0	1 (0.3)	0	33

*: Metaphase plate with one or more than one aberrations considered as one metaphase plate with aberrations

@: values are the sum of two replicates and values in parenthesis represent %  

Cs: Chromosome type; Ct: Chromatid type                                 

Note: There were no incidences of polyploidy and endoreduplicated cells

Summary Results of Chromosomal Aberration Assay - Experiment 3

Refer Appendices 3 and 6

Treatment (µg/mL)	No. of metaphases scored	No. (%) of metaphases with aberrations^@						Total No. (%) of aberrant metaphases^*		Cell Growth Inhibition (%)
		Gaps		Breaks		Exchanges		Including Gaps	Excluding Gaps
		Cs	Ct	Cs	Ct	Cs	Ct
DMSO	300	0	0	0	0	0	0	0	0	0
250	300	0	0	0	0	0	0	0	0	31
500	300	2 (0.7)	0	0	0	0	0	2 (0.7)	0	36
1000	300	1 (0.3)	0	0	0	0	0	1 (0.3)	0	41
EMS 600	300	0	1 (0.3)	42 (14.0)	54 (18.0)	46 (15.3)	18 (6.0)	147 (49.0)	+ 147 (49.0)	49

*: Metaphase plate with one or more than one aberrations considered as one metaphase plate with aberrations

@: values are the sum of two replicates and values in parenthesis represent %  

Cs: Chromosome type; Ct: Chromatid type; EMS: Ethyl methanesulfonate; +: Significantly higher than control (p <0.05) by Fischer exact test

Note: There were no incidences of polyploidy and endoreduplicated cells

 

Conclusions:

The test item was not clastogenic in CHO-K1 cells at the tested concentration (up to 1000 µg/mL) and under the conditions of testing employed.

Executive summary:

The clastogenic potential of the test item 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 was formed a free flowing suspension in dimethyl sulfoxide (DMSO) at 100 mg/mL.

 

In a preliminary cytotoxicity test for the selection of test concentrations for the chromosomal aberration assay, CHO-K1 cells exposed to the test item, did not exhibit the required level of cytotoxicity as RICC at the highest tested concentration of 1000 µg/mL, compared to the DMSO control, both in the presence (3-hour exposure) and absence of metabolic activation (3 and 21-hour exposure). Slight precipitation of the test item in the test medium was observed at and above 256 µg/mL. The test item did not cause any appreciable changes in the pH and osmolality of the test medium. Based on these observations, a maximum of 1000 µg/mL was tested in all the three experiments of the chromosomal aberration assay.

 

In the definitive chromosomal aberration assay, CHO-K1 cells were exposed to the test item in duplicate for at the concentrations of 250, 500 and 1000mg/mL in all the three experiments of the chromosomal aberration assay. Concurrent vehicle (DMSO) and positive controls (cyclophosphamide monohydrate in the presence of metabolic activation and ethyl methanesulfonate in the absence of metabolic activation) were also tested in duplicate. In each case, the cells in C-metaphase were harvested at 21 hours after the start of the treatment and slides were prepared for chromosomal analysis.

 

At the respective highest concentrations tested, the reduction in cell growth as RICC was 32, 33 and 41 % in experiments 1, 2 and 3, respectively, compared to the vehicle control.

 

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

There was no evidence of 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 was not clastogenic at the concentrations tested and under the conditions of testing.

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

according to OECD and GLP guidelines

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

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: American Type Culture Collection, P.O.Box 1549, Manassas, VA 20108
USA

- Suitability of cells: 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.

- Cell cycle length, doubling time or proliferation index: Chinese Hamster (Cricetulus griseus) ovary cell line CHO-K1, (ATCC CCL-61, Lot 4765275) 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).

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Ham’s F-12 medium supplemented with sodium bicarbonate, antibiotics, and L-glutamine was the basic medium.
Basic medium supplemented with 10% fetal bovine serum (FBS) was the complete medium and was used for the growth and multiplication of cells as well as in detaching and diluting the cells.
Basic medium supplemented with 5% fetal bovine serum (FBS) was the treatment medium and was used for target cell exposure to the test item and controls.
Cloning medium was basic medium supplemented with 20 % FBS and was used for the determination of cell viability or plating/cloning efficiency.
Selective medium was basic medium supplemented with 20 % FBS and the selective agent 6-Thioguanine (6-TG) at 35 µM and was used for the selection of mutants.
Dulbecco’s Phosphate buffered saline (PBS) (pH: 7.4) Trypsin: EDTA solution

- Properly maintained: [yes]
- Periodically checked for Mycoplasma contamination: [yes]
- Periodically checked for karyotype stability: [yes)

Metabolic activation:

with and without

Metabolic activation system:

S9 homogenate was prepared from male Wistar rats induced with a single intra-peritoneal injection of Aroclor 1254 (0.7 mL/rat ready to use solution), 5 days prior to sacrifice and stored in a deep freezer maintained at -68 to -86 ºC.

Test concentrations with justification for top dose:

Based on the results of the preliminary cytotoxicity test, the following test concentrations were selected for testing in the gene mutation test:
Experiments 1 and 2 (Presence and Absence of Metabolic Activation, respectively)

A) 125 B) 250 C) 500 and D) 1000 µg/mL (factor of 2)

Vehicle / solvent:

One hundred fifty microliters (150 L) of Dimethyl sulphoxide (DMSO) was used per 15 mL of treatment medium as the vehicle control in each of the experiments.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; Exponentially growing CHO-KI cells were plated in two replicates and each replicate has two flasks containing 15 mL of complete medium at a density of approximately 4.5 x 106 cells / 75 cm2 flask and incubated for approximately 24 hours.
Two parallel cultures were also kept along with the vehicle control and treatment groups. Cell counts were made from these cultures at the 0-hour treatment to obtain the baseline cell count for estimation of Relative Survival (RS).

- Cell density at seeding (if applicable): 4.5 x 10 6 cells / 75 cm 2

DURATION
All test item and positive control concentrations were prepared immediately before use in sterile test tubes. The target cells were exposed to the vehicle, positive control and various concentrations of the test item for 3 hours in the presence and absence of metabolic activation.
The medium from each target cells flask was removed by aspiration and replaced with 13.5 mL and 15 mL of treatment medium for the experiment in the presence and absence of metabolic activation, respectively. For the experiment incorporating metabolic activation, 1.5 mL of S9 mix was added to give a final concentration of 1 % S9 (v/v) in the test medium.
One hundred fifty microliters (150 µL) each of the vehicle control, the positive control, dilution of the test item were transferred to respective flasks and gently mixed and kept for incubation to expose the cells to treatment.

- Exposure duration: 3 hours
- Expression time (cells in growth medium): 9 days
- Selection time (if incubation with a selection agent): 10 days

SELECTION AGENT (mutation assays): Replicate cultures from controls and each treatment level were trypsinized, and the cells suspended in 10 mL complete medium, pooled, and counted using a hemocytometer.
For selection of the 6-Thioguanine (6-TG) resistant phenotype, cells from each of the replicate cultures were plated in to 5 flasks at a density of approximately 2 x 105 cells/25 cm2 flask (total of 106 cells/replicate) in selective medium and incubated for 10 days.
For cloning efficiency determination at the time of selection, cells from each of the replicate cultures were plated approximately at 200 cells/25 cm2 flask in triplicate in cloning medium and incubated for 10 days.

STAIN (for cytogenetic assays): The colonies were stained with 0.5 % methylene blue and counted for both cloning efficiency and mutant selection after 10 days of incubation.

NUMBER OF REPLICATIONS: Two

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

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.

Statistics:

A power transformation procedure (Snee and Irr, 1981) was used with which, the observed mutant frequency was transformed using the formula:

Y = (X + A) B

where,
Y = transformed mutant frequency
X = observed mutant frequency
and A, B = constants.

Statistical analysis of the experimental data was carried out using validated copies of SYSTAT Statistical package version 12.0. In cases where analysis of variance was significant at p < 0.05, a Dunnett’s test was conducted, comparing each treatment group and the positive control to the vehicle control (p < 0.05).

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

 Summary Results of the Gene Mutation Assay in the Presence of Metabolic Activation (Experiment 1)

Treatment µg/mL	Mutation Assay Flasks						Cloning Efficiency of Mutant Colonies	Cloning Efficiency Flasks				6-TG Mutants per 10^6Clonable Cells (MF)
	TG Colonies/Flask							No. of Colonies/Flask
	1	2	3	4	5	Total		1	2	3	CE*
DMSO	1	2	2	2	2	17	0.0000085	185	181	189	0.918	9.26
	1	2	2	1	2			181	185	180
125	1	1	2	1	2	14	0.000007	187	180	182	0.918	7.63
	3	1	1	1	1			184	187	181
250	3	1	1	1	1	14	0.000007	180	179	184	0.904	7.74
	2	1	2	1	1			181	181	180
500	2	1	1	1	1	12	0.000006	179	180	178	0.901	6.66
	2	1	1	1	1			178	180	186
1000	2	0	3	2	2	18	0.000009	177	179	178	0.893	10.08
	2	1	2	2	2			175	180	182
3-MCA	29	33	39	29	35	337	0.000169	139	143	147	0.728	232.14^+
	35	30	35	37	35			144	151	149

Positive Control: 8 µg/mL 3-MCA             Vehicle Control: DMSO                                                 CE: Cloning Efficiency               MF: Mutant Frequency

* calculated from the mean values of the replicates of each group and rounded off to three decimal places

  Mutant frequency of 6-TG mutants is significantly higher than the concurrent vehicle control value (p < 0.05)

 

CE	=	Total No. of colonies		MF	=	CE of mutant colonies in selective medium	x 10^6
		No. of cells plated				CE in non-selective medium

 

Summary Results of the Gene Mutation Assay in the Absence of Metabolic Activation (Experiment 2)

Treatment µg/mL	Mutation Assay Flasks						Cloning Efficiency of Mutant Colonies	Cloning Efficiency Flasks				6-TG Mutants per 10^6Clonable Cells (MF)
	TG Colonies/Flask							No. of Colonies/Flask
	1	2	3	4	5	Total		1	2	3	CE*
DMSO	2	1	3	1	2	17	0.0000085	184	183	185	0.923	9.21
	1	2	1	2	2			182	191	183
125	2	2	1	2	1	16	0.000008	184	189	181	0.925	8.65
	2	1	1	2	2			185	187	184
250	2	1	1	1	1	13	0.0000065	170	165	164	0.858	7.58
	1	2	2	1	1			175	177	178
500	1	2	2	2	2	16	0.000008	156	160	165	0.818	9.78
	1	2	2	1	1			165	165	170
1000	1	1	1	2	2	13	0.0000065	152	146	139	0.723	8.99
	1	1	1	1	2			147	140	143

Vehicle Control: DMSO                             CE: Cloning Efficiency                    MF: Mutant Frequency                                                              

* calculated from the mean values of the replicates of each group and rounded off to three decimal places

 

 

CE	=	Total No. of colonies		MF	=	CE of mutant colonies in selective medium	x 10^6
		No. of cells plated				CE in non-selective medium

 

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 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 free flowing suspension in dimethyl sulphoxide (DMSO) at 100 mg/mL.

 

In a preliminary cytotoxicity test for the selection of test concentrations for the gene mutation assay, the Relative Survival was 48 and 57 % at the 1000 µg/mL, in the presence and absence of metabolic activation, respectively. There was slight precipitation of the test item in the test medium at and above 256 µg/mL. There was no appreciable change in the pH and osmolality of test medium. Based on these observations a maximum of 1000 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 125, 250, 500 and 1000 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

C.I. Pigment Orange 68 did not reveal any mutagenic effect in the

• bacterial reverse mutation assays (AMES) in the presence or absence of metabolic activation,
• in the in vitro gene mutation study in mammalian cells (HPRT) in the presence or absence of metabolic activation, and
• in the mammalian cell chromosome aberration test in the presence or absence of metabolic activation.

Based on these findings the test item has not to be classified as germ cell mutagen according to Regulation (EC) No 1272/2008.