Calcium bis[4-[[3-[[2-hydroxy-3-[[(4-methoxyphenyl)amino]carbonyl]-1-naphthyl]azo]-4-methylbenzoyl]amino]benzenesulphonate]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item did not exert mutagenic activity in the reverse bacterial mutation assay (plate incorporation assay and pre-incubation assay according to Prival) with and without metabolic activation.

The test substance did not increase the mutant frequency at the HPRT locus in Chinese hamster V79 cells and is therefore considered to be non-mutagenic under the conditions of the test.

It was concluded that the test item, was not clastogenic in CHO cells at the tested concentrations and under the conditions of testing employed.

Link to relevant study records

Reference

Reference 1

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

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media:supplemented MEM
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

liver S9 mix from phenobarbital/beta-naphthoflavone pretreated Wistar rats

Test concentrations with justification for top dose:

without S9 mix:
Experiment I: 1.3, 2.7, 5.3, 10.7, 21.3 and 170.0 µg/ml
Experiment II: 1.3, 2.7, 5.3, 10.7, 21.3 and 170.0 µg/ml

With S9 mix:
Experiment I: 1.3, 2.7, 5.3, 10.7, 21.3 and 170.0 µg/ml

Not all test groups were evaluated (for evaluation points see executive summary)

Vehicle / solvent:

- DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Experiment I: 4 h, experiment II: 24 h
- Expression time (cells in growth medium): 24 h
- Selection time (if incubation with a selection agent): 8 days
- Fixation time (start of exposure up to fixation or harvest of cells): 3+7+8 days

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS: 2 experiments, treatments performed in duplicate, both with and without metabolic activation

NUMBER OF CELLS EVALUATED: colonies with more than 50 cells

DETERMINATION OF CYTOTOXICITY
- Method: colony formation

Evaluation criteria:

A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration- related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontanous mutation frequency at least at one of the concentrations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the corresponding negative control data. If there is by chance a low spontanous mutation rate in the range normally found (0.5-31.8 mutants per 10 to the power of 6 cells) a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of negative and solvent controls within all experiments of this study was also taken into considration.

Statistics:

A linear regression was performed to assess a possible dose dependent increase of mutant frequencies.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: from 10.7 µg/ml with and without metabolic activation in both experiments.

RANGE-FINDING/SCREENING STUDIES:
A pre-test was performed with concentrations ranging from 1.3 to 170.0 µg/ml

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results:
negative

The test substance did not increase the mutant frequency at the HPRT locus in Chinese hamster V79 cells and is therefore considered to be non-mutagenic under the conditions of the test.

Executive summary:

The test item was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster according to OECD TG 476. Chinese hamster V79 cells were treated with the test substance in the following concentrations:

without S9 mix:

Experiment I: 1.3, 2.7, 5.3, 10.7, 21.3 and 170.0 µg/ml

Experiment II: 1.3, 2.7, 5.3, 10.7, 21.3 and 170.0 µg/ml

With S9 mix:

Experiment I: 1.3, 2.7, 5.3, 10.7, 21.3 and 170.0 µg/ml

Marked in bold: cultures which were not continued.

In experiment I and IA the treatment period was 4 h with and without metabolic activation. The second experiment was performed in the absence of metabolic activation with a treatment time of 24 h.

A precipitation of the test substance was noted in both experiments at 10.7 and 170 µg/ml with and without metabolic activation. The solvent control gave mutant frequencies within the normal range. The positive controls gave marked increases in the mutant frequency, 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.

Reference 2

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Read across from structural analogue

Justification for type of information:

see Rationale and Justification for the Analogue Read-Across Approach - Nanoforms and Bulk Forms (Chapter 13)

Reason / purpose for cross-reference:

read-across source

Statistics:

A

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: from 10.7 µg/ml with and without metabolic activation in both experiments.

RANGE-FINDING/SCREENING STUDIES:
A pre-test was performed with concentrations ranging from 1.3 to 170.0 µg/ml

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results:
negative

The test substance did not increase the mutant frequency at the HPRT locus in Chinese hamster V79 cells and is therefore considered to be non-mutagenic under the conditions of the test.

Executive summary:

The test item was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster according to OECD TG 476. Chinese hamster V79 cells were treated with the test substance in the following concentrations:

without S9 mix:

Experiment I: 1.3, 2.7, 5.3, 10.7, 21.3 and 170.0 µg/ml

Experiment II: 1.3, 2.7, 5.3, 10.7, 21.3 and 170.0 µg/ml

With S9 mix:

Experiment I: 1.3, 2.7, 5.3, 10.7, 21.3 and 170.0 µg/ml

Marked in bold: cultures which were not continued.

In experiment I and IA the treatment period was 4 h with and without metabolic activation. The second experiment was performed in the absence of metabolic activation with a treatment time of 24 h.

A precipitation of the test substance was noted in both experiments at 10.7 and 170 µg/ml with and without metabolic activation. The solvent control gave mutant frequencies within the normal range. The positive controls gave marked increases in the mutant frequency, 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.

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:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant guideline study (OECD TG471) with Prival modification for azo-dyes

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Directive 2000/32/EC, L1362000, Annex 4D

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 or hamster liver S9

Test concentrations with justification for top dose:

Experiment I (Plate incorporation method) and Experiment II (Pre-incubation method): 0, 3, 10, 33, 100, 333, 1000, 2500, 5000 µg/plate

Vehicle / solvent:

- Vehicle/ Solvent used: DMSO
- Justification for choice of solvent/ vehicle: solubility properties and its relative non-toxicity to the bacteria

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide (TA 1535 and TA 100), 4-Nitro-o-phenylene-diamine (TA 1537 and TA 98), methyl methane sulfonate (WP2 uvrA)

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation (rat liver S9 mix)

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 and WP2 uvrA), congo red (TA98)

Remarks:

with metabolic activation (hamster liver S9 mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Experiment I: plate incorporation method without and with induced rat liver S9 mix (induction with phenobarbital/beta-naphthoflavone)
Experiment II: pre-incubation method without and with non-induced hamster liver S9 mix.

DURATION:
-preincubation period (experimentII): 30 °C, for 30 min
- exposure duration: 48 h at 37 °C in the dark

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

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, WP2 uvrA) or thrice (strains TA 1535, TA 1537) the colony count of the corresponding solvent colony is observed.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the thresshold is regarded as an indication of mutagenic potential if reproduced in an independent second experiment. however, whenever the colony counts remain within the historical range of negative annd solvent controls such as an increase is not considered biologically relevant.

Statistics:

Arithmetic mean, standard deviation and the ratio of treated versus solvent 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

Remarks:

Only strain TA 1537 in experiment I with metabolic activation and experiment II without metabolic activation and strain TA 1535 in experiment II with metabolic activation showed some minor toxic effects at 5000 mg/plate (reduced no of revertants).

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: occurred at concentrations of 333 µg/plate and more (Experiment I and Experiment II with and without metabolic activation). Nevertheless undissolved particles had no influence on the data recording.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results:
negative

The test item did not exert mutagenic activity in the reverse bacterial mutation assay (plate incorporation assay and pre-incubation assay according to Prival) 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 0, 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 same concentrations.

The test item did not reveal any mutagenic activity under the conditions tested. The appropriate reference mutagenes showed distinct positive mutagenic effects.

Reference 4

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: Ham's F-12
- 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

Test concentrations with justification for top dose:

Final test concentration in the medium:

a) 14 µg/mL
b) 42 µg/mL
c) 126 µg/mL

(factor of 3)

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Remarks:

Ethyl methanesulphonate (CAS No. 62-50-0) (EMS), Cyclophosphamide monohydrate (CAS No. 6055-19-2) (CPA)

Details on test system and experimental conditions:

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 10 to 14 hours was used.

Batch No. 3 of this CHO-K1 cell line was tested for the absence of mycoplasma contamination at Mycoplasma Laboratory, Statens Serum Institut, Artillerivej 5, Copenhagen S, Denmark and certified free of mycoplasma contamination on 11 August 2014.

Cells were grown in T-25 and T-75 cm2 flasks for the preliminary cytotoxicity and chromosome aberration assay, respectively, at 37 ± 1 °C in a carbon dioxide incubator (5 ± 0.2 % CO2 in air).

Cell cultures were exposed to the test item for 3 hours both in the presence and absence of exogenous metabolic activation and for 21 hours in the absence of metabolic activation. Following exposure of cell cultures to the test item, they were treated with Colchicine to arrest the cells in a metaphase-like stage of mitosis (c-metaphase). Cells were then harvested and chromosome preparations made. Preparations were stained and metaphases were analyzed for chromosomal aberrations.

Evaluation criteria:

Metaphases of the three concentrations of the test items, the positive and vehicle controls cultures were scored.

Wherever possible, 300 metaphases whose centromere number at best varies by ± 2 from the modal number of 20 were evaluated among the
2 replicates. The chromosome number was recorded for all cells analyzed and the vernier readings of only aberrant cells were recorded. Aberrations, if any were recorded as chromatid / chromosome gaps or breaks and exchange figures. Since gaps are not considered as true aberration, the results are presented as metaphases with aberrations including gaps and excluding gaps.

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, numerical aberration (Poly) were analyzed. Pooled data from each test concentration and the positive control were compared with the vehicle control using Fisher 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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

S-9 Characterization

a) Sterility Check

The S-9 homogenate was found to be sterile.

b) Metabolic Activation

The S-9 homogenate was characterized for its ability to metabolize the promutagens 2-aminoanthracene and benzo(a)pyrene to mutagens using Salmonella typhimurium TA100 strain and the S-9 homogenate was found to be active.

c) Protein Content

The protein content of the S-9 homogenate was 30 mg/mL and was found to be satisfactory.

 

Solubility Test and Justification for the Selection of Vehicle

The test item formed a free flowing suspension in DMSO at 50 mg/mL.

DMSO is one of the organic vehicles compatible with this test system. Hence, based on the results of solubility test, DMSO was selected as the vehicle of choice to prepare the stock and dilutions of the test item.

 

Cytotoxicity Test and Justification for the Selection of Test Doses

The results of pH assessment of test solutions

Treatment (µg/mL)	pH at the beginning of exposure to treatment		pH at the end of exposure to treatment
	With S-9	Without S-9	With S-9	Without S-9
DMSO	7.04	7.19	7.08	7.31
2.5	7.09	7.21	7.16	7.25
5	7.07	7.21	7.15	7.28
10	7.07	7.23	7.14	7.29
20	7.08	7.24	7.11	7.31
40	7.10	7.25	7.14	7.32
80	7.10	7.25	7.14	7.33
160	7.13	7.26	7.16	7.31
320	7.13	7.26	7.16	7.33
500	7.17	7.28	7.13	7.34

 

The results of osmolality assessment of test solutions

Treatment (g/mL)	Osmolality at the beginning of exposure to treatment (OSMOL/kg)		Osmolality at the end of exposure to treatment (OSMOL/kg)
	With S-9	Without S-9	With S-9	Without S-9
DMSO	0.431	0.415	0.422	0.429
80	-	-	0.420	0.438
160	-	-	0.411	0.423
500	0.303	0.397	0.413	0.421

 

The results of the cytotoxicity test

Treat-ment (µg/mL)	Presence of metabolic activation (3-hour exposure)			Absence of metabolic activation (3-hour exposure)			Absence of metabolic activation (21-hour exposure)
	Final – initial cell count (1x10^6/ flask)	Cell growth index RICC (%)	Cell growth inhibition (%)	Final – initial cell count (1x10^6/ flask)	Cell growth index RICC (%)	Cell growth inhibition (%)	Final – initial cell count (1x10^6/ flask)	Cell growth index RICC (%)	Cell growth inhibition (%)
DMSO (150 μL)	2.850	100	0	3.100	100	0	2.450	100	0
2.5	2.500	88	12	2.900	94	6	2.250	92	8
5	2.350	82	18	2.700	87	13	2.100	86	14
10	2.000	70	30	2.650	85	15	1.875	77	23
20	1.825	64	36	2.450	79	21	1.900	78	22
40	1.625	57	43	2.150	69	31	1.600	65	35
80	1.375	48	52	1.550	50	50	1.150	47	53
160	1.125	39	61	0.750	24	76	0.400	16	84
320	1.075	38	62	- 0.175	0	-	- 0.225	0	-
500	0.400	14	86	- 0.45	0	-	- 0.600	0	-

 

Based on these observations, for all the 3 experiments of the chromosomal aberration assay, a maximum of 126 μg/mL was tested.

Chromosomal Aberration Assay

Experiment 1

At the highest concentration tested (126 μg/mL), the reduction in the cell growth was 56 % compared to the vehicle control.

The incidence of aberrations in the vehicle control was within the range of the in-house historical control data.

The incidence of aberrant metaphases excluding gaps was statistically comparable to the vehicle control value at all the concentrations tested.

No incidences of polyploidy, endoreduplication and pulverization observed.

The positive control, cyclophosphamide monohydrate caused a statistically significant increase in the aberrant metaphases excluding gaps.

 

Experiment 2

At the highest concentration tested (126 μg/mL), the reduction in the cell growth was 57 % compared to the vehicle control.

The incidence of aberrations in the vehicle control was within the range of the in-house historical control data.

The incidence of aberrant metaphases excluding gaps was statistically comparable to the vehicle control value at all the concentrations tested.

No incidences of polyploidy, endoreduplication and pulverization observed.

 

Experiment 3

At the highest concentration tested (126 μg/mL), the reduction in the cell growth was 59 % compared to the respective vehicle control.

The incidence of aberrations in the vehicle control was within the range of the in-house historical control data.

The incidence of aberrant metaphases excluding gaps was statistically comparable to the vehicle control value at all the three test concentrations.

No incidences of polyploidy, endoreduplication and pulverization observed.

 

The positive control, ethyl methanesulphonate caused a statistically significant increase in the aberrant metaphases excluding gaps.

CONCLUSIONS

It was concluded that the test item, was not clastogenic in CHO cells at the tested concentrations and under the conditions of testing employed.

Conclusions:

It was concluded that the test item, was not clastogenic in CHO cells at the tested concentrations 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) 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 chromosomal aberration assay. Chromosomal 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 DMSO at 50 mg/mL.

 

In a preliminary cytotoxicity test for the selection of test concentrations for the chromosomal aberration assay, the test item showed required level of cytotoxicity as RICC compared to the vehicle control between 80 and 160 µg/mL test concentrations in the presence and absence of metabolic activation with 3-hour exposure as well as in the absence of metabolic activation with 21-hour exposure. The test item precipitated in the test medium and above 160 µg/mL but did not cause any appreciable change in the Osmolality of test medium. Based on these observations, a maximum of 126 µg/mL was tested in all the three experiments of the chromosomal aberration assay.

 

In the chromosomal aberration assay, CHO-K1 cells were exposed to the test item in duplicate for at the concentrations of 14, 42 and 126 mg/mL (experiments 1 and 2 for 3 hours in the presence and absence of metabolic activation, respectively and experiment 3 for 21 hours in the absence of metabolic activation). 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 highest concentration tested, the reduction in cell growth as RICC was 56, 57 and 59 % 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 control 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. There was also no incidence of polyploid cells indicating that the test item did not induce numerical chromosomal aberrations. 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Discussion

Pigment Red 247 did not induce gene mutations by frameshifts or base-pair substitutions in the genome of the strains used when tested in bacterial reverse mutation assays (Ames tests with and without Prival modification, test strains: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2) with and without metabolic activation (rat liver S9 mix, and hamster liver S9 mix) at concentrations up to 5000 µg/plate.

 

Pigment Red 247 didn't cause structural chromosomal aberrations when tested in the in vitro mammalian chromosome aberration test using Chinese hamster ovary cell line CHO-K1, neither with nor without metabolic activation at concentrations up to 126 µg/ml.

 

Pigments with similar chemical structure (PR 112, PR 146, PR 147, PR 170) were not genotoxic in the mammalian cell gene (HPRT) mutation test in V79 Chinese Hamster cells when tested with and without rat liver S9 metabolic activation.

 

In conclusion the test items did not induce gene mutations in bacteria or mammalian cells and didn't cause structural chromosomal aberrations in mammalian cells.

Justification for classification or non-classification

No classification

None of the in vitro findings points to genotoxic properties ot the items tested. Thus, the substance subject to registration does not meet criteria for classification according to REGULATION (EC) No 1272/2008.