Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance was found not mutagenic in Salmonella typhimurium and Escherichia coli (Ames test). No potential to cause mutations and damage to chromosomes in mammalian cells is expected based on data obtained for other category members.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 Dec 2011 - 19 Dec 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Principles of method if other than guideline:
The Prival preincubation test is a modification of the standard Ames reverse mutation assay, in which flavin mononucleotide (FMN), liver S9 mix from uninduced hamsters and a preincubation step are used to facilitate azo reduction and the detection of the resulting mutagenic aromatic amines.
GLP compliance:
yes (incl. certificate)
Remarks:
BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany
Type of assay:
bacterial reverse mutation assay
Target gene:
his, trp
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver S-9 mix and uninduced hamster liver S-9 mix
Test concentrations with justification for top dose:
33 μg - 5 000 μg/plate (SPT)
33 μg - 5 000 μg/plate (PIT)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: with rat S9 (SPT): 2-aminoanthracene (all strains) - with hamster S9 (PIT): 2-aminoanthracene (all strains), Congo red (TA98), benzidine (TA98)
Remarks:
with S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-methyl-N'-nitro-N-nitrosoguanidine (TA1535, TA100), 4-nitro-o-phenylendiamine (TA98), 9-aminoacridine (TA1537), 4-nitroquinoline-N-oxide (E. coli WP2 uvrA)
Remarks:
without S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); Prival preincubation
The Prival preincubation test is a modification of the standard Ames reverse mutation assay, in which flavin mononucleotide (FMN), liver S9 mix from uninduced hamsters and a preincubation step are used to facilitate azo reduction and the detection of the resulting mutagenic aromatic amines

DURATION
- Preincubation period: 30 min
- Exposure duration: 48 – 72 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
The test chemical is considered positive in this assay if the following criteria are met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.

A test substance is generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.
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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Test substance precipitation was found from 333 μg/plate onward with and without S9 mix.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A weak bacteriotoxic effect (slight decrease in the number of his+ revertants) was occasionally observed in the Ames standard plate test depending on the strain and test conditions from 2500 μg/plate onward. In the Prival preincubation assay weak bacteriotoxicity (slight decrease in the number of his+ or trp+ revertants) was observed depending on the strain and test conditions at 5000 μg/plate.

According to the results of the present study, the test substance did not lead to a biologically relevant increase in the number of revertant colonies either without S9 mix or after adding a metabolizing system in two experiments carried out independently of each other (Ames standard plate test and Prival preincubation assay). Besides, the results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study. In this study with and without S9 mix, the number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain. In addition, the positive control substances both with and without S9 mix induced a significant

increase in the number of revertant colonies within the range of the historical positive control data or above.

Results of Experiment I (Standard Plate Test with and without rat liver S-9 mix):

TA98 TA100 TA1535 TA1537 WP2 uvrA
concentration -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
DMSO 20 22 79 84 11 14 6 7 47 48
33 19 23 76 82 11 15 6 7 45 51
100 19 24 79 78 13 15 6 8 44 53
333 21 21 87 81 10 15 6 6 43 49
1000 20 25 86 75 12 16 6 8 19 49
2500 17 19 74 78 11 14 5 5 46 52
5000 20 16 73 70 14 14 4 5 47 47
MNNG 675 1176
2-AA 864 790 174 123 237
AAC 363
NOPD 531
4-NQO 608

Controls:

MNNG: N-methyl-N'-nitro-N-nitrosoguanidine (5 µg/plate)

2 -AA: 2-aminoanthracene (2.5 µg/plate for TA 98, 100, 1535, 1537; 60 µg/plate for WP2 uvrA)

AAC: 9-aminoacridine (100 µg/plate)

NOPD: 4-nitro-o-phenylendiamnine (10 µg/plate)

4 -NQO: 4-nitroquinoline-N-oxide (5 µg/plate)

Results of Experiment II (Pre-incubation Test with and without hamster liver S-9 mix):

TA98 TA100 TA1535 TA1537 WP2 uvrA
concentration -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
DMSO 28 40 120 133 16 18 8 10 51 51
33 29 36 125 1217 16 17 8 11 45 52
100 26 38 113 130 16 16 8 13 49 46
333 26 31 123 130 18 20 8 9 50 46
1000 26 34 119 117 16 17 6 10 50 47
2500 26 31 118 115 19 16 7 9 39 57
5000 21 37 105 102 14 13 7 10 49 38
MNNG 854 740
2-AA 852 1695 742 142 252
AAC 478
NOPD 708
4-NQO 235
CONGOR. 639
BENZID. 655

Controls:

MNNG: N-methyl-N'-nitro-N-nitrosoguanidine (5 µg/plate)

2 -AA: 2-aminoanthracene (10 µg/plate)

AAC: 9-aminoacridine (100 µg/plate)

NOPD: 4-nitro-o-phenylendiamnine (10 µg/plate)

4 -NQO: 4-nitroquinoline-N-oxide (5 µg/plate)

CONGOR: Congo red (0.3 µmol/plate)

benzid: benizidine (0.3 µmol/plate)

Conclusions:
Under the experimental conditions chosen here, it is concluded that the test article is not a mutagenic substance in the bacterial reverse mutation test (Ames standard plate test and Prival preincubation assay) in the absence and the presence of metabolic activation.
Executive summary:

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of Salmonella typhimurium strains TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA in a reverse mutation assay (Ames standard plate test and Prival preincubation test). The Prival preincubation test is a modification of the standard Ames reverse mutation assay, in which flavin mononucleotide (FMN), liver S9 mix from uninduced hamsters and a preincubation step are used to facilitate azo reduction and the detection of the resulting mutagenic aromatic amines .This test is therefore the most appropriate method for the investigation of azo-dyes and diazo compounds such as the test compound. Both assays were performed in the presence and absence of a metabolic activation system at a concentration range of 33 – 5000 µg/plate. Precipitation of the test substance was found from a concentration of 333 μg/plate onward with and without S9 mix. A weak bacteriotoxic effect (slight decrease in the number of his+ revertants) was occasionally observed in the Ames standard plate test depending on the strain and test conditions from 2500 μg/plate onward. In the Prival preincubation assay weak bacteriotoxicity (slight decrease in the number of his+ or trp+ revertants) was observed depending on the strain and test conditions at 5000 μg/plate. According to the results of the present study, the test substance did not lead to a biologically relevant increase in the number of revertant colonies either without S9 mix or after adding a metabolizing system in two experiments carried out independently of each other (Ames standard plate test and Prival preincubation assay). Besides, the results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study. In this study with and without S9 mix, the number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain. In addition, the positive control substances both with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data or above. Thus, under the experimental conditions of this study, the test substance is not mutagenic in the Ames standard plate test and in the Prival preincubation test in the absence and the presence of metabolic activation.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 20, 2011 - December 15, 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
GLP compliance:
yes (incl. certificate)
Remarks:
Harlan Cytotest Cell Research GmbH
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT (hypoxanthine-guanine phosphoribosyl transferase)
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM (minimal essential medium) containing Hank’s salts, neomycin (5 Pg/mL) and amphotericin B (1 %). For the selection of mutant cells the complete medium was supplemented with 11 Pg/mL 6-thioguanine. All cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2.
- 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:
Phenobarbital/ß-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Without S9 mix: 7.0; 14.1; 28.1; 56.3; 112.5; 225.0 µg/ml
With S9 mix: 7.0; 14.1; 28.1; 56.3; 112.5; 225.0 µg/ml
In experiment I and II the cultures at the maximum concentration with and without metabolic activation were not continued to avoid evaluation of too many precipitating concentrations
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle:The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures. The final concentration of acetone in the culture medium was 0.5 % (v/v).
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: with S9 mix: DMBA; 7,12-dimethylbenz(a)anthracene, 1.1 Pg/mL = 4.3 µM; without S9 mix: EMS; ethylmethane sulfonate, 0.150 mg/mL = 1.2 mM
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

Approximately 1.5×10E6 (single culture) and 5×10E2 cells (in duplicate) were seeded in plastic culture flasks. After 24 hours the medium was replaced with serum-free medium containing the test item, either without S9 mix or with 50 µL/mL S9 mix. Concurrent solvent and positive controls were treated in parallel. After 4 hours this medium was replaced with complete medium following two washing steps with "saline G". In the second experiment the cells were exposed to the test item for 24 hours in complete medium, supplemented with 10 % FBS, in the absence of metabolic activation.
The colonies used to determine the cloning efficiency (survival) were fixed and stained approximately 7 days after treatment as described below. Three or four days after treatment 1.5×10E6 cells per experimental point were subcultivated in 175 cm² flasks containing 30 mL medium. Following the expression time of 7 days five 80 cm² cell culture flasks were seeded with about 3 - 5×10E5 cells each in medium containing 6-TG. Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability. The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 for about 8 days. The colonies were stained with 10 % methylene blue in 0.01 % KOH solution. The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

PRE-TEST ON TOXICITY
A pre-test was performed in order to determine the concentration range for the mutagenicity experiments. In this pre-test the colony forming ability of approximately 500 single cells (duplicate cultures per concentration level) after treatment with the test item was observed and compared to the controls. Toxicity of the test item is indicated by a reduction of the cloning efficiency (CE). Based on the solubility properties of the test item the range finding pre-experiment test was performed using a concentration range of 14.1 to 1800 µg/mL to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24 hours treatment) of metabolic activation. No relevant cytotoxic effect indicated by a relative suspension growth below 50 was noted up to the maximum concentration of 1800 Pg/mL with and without metabolic activation following 4 and 24 hours treatment.
The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal to the test item. Precipitation occurred at 112.5 µg/mL and above in the presence and absence of metabolic activation following 4 and 24 hours treatment.
Based on the occurrence of precipitation in the pre-experiment, the individual concentrations of the main experiments were selected. The individual concentrations were spaced by a factor of 2.
Evaluation criteria:
The gene mutation assay is considered acceptable if it meets the following criteria:
- The numbers of mutant colonies per 10E6 cells found in the solvent controls fall within the laboratory historical control data.
- The positive control substances should produce a significant increase in mutant colony frequencies.
- The cloning efficiency II (absolute value) of the solvent controls should exceed 50 %.

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 spontaneous 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 solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.
Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance was considered together.
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 applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In both main experiments precipitation was observed at 56.3 Pg/mL and above in the presence and absence of metabolic activation.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No relevant cytotoxic effect indicated by a relative suspension growth below 50 was noted up to the maximum concentration of 1800 µg/mL with and without metabolic activation following 4 and 24 hours treatment.

No relevant and reproducible increase in mutant colony numbers/106 cells was observed in the main experiments up to the maximum concentration. The mutant frequency remained well within the historical range of solvent controls. An increase of the induction factor exceeding or reaching the threshold of three times the mutation frequency of the corresponding solvent control was observed in the second culture of the first experiment with metabolic activation at the lowest concentration of 7.0 µg/mL and at 28.1 µg/mL. However, the increase was based on a rather low mutation frequency of the solvent control of just 3.4 colonies per 106 cells. Furthermore, the effect was not reproduced in the parallel culture under identical experimental conditions. Therefore, the increase of the induction factor was judged as biologically irrelevant fluctuation.

Summary of results:

concentration (µg/ml) P S9 Mix relative cloning efficiency I (%) relative cell density (%) relative cloning efficiency II (%) mutant colonies / 106cells induction factor relative cloning efficiency I (%) relative cell density (%) relative cloning efficiency II (%) mutant colonies / 106cells induction factor
Experiment I / 4h treatment culture I culture II
solvent control (acetone) - 100 100 100 20.8 1 100 100 100 17.9 1
positive control (EMS) 150 - 71.8 108.9 98.1 116 5.6 85.9 139.1 81.8 113.3 6.3
test item 7 - 96.9 109.1 86 30.1 1.4 99.3 120.9 88.6 6.4 0.4
test item 14.1 - 96.9 123.5 112.1 11.9 0.6 97.1 124.5 84 22.6 0.3
test item 28.1 - 89.1 113.2 88.5 14.8 0.7 95.1 154.7 82.9 15.2 0.8
test item 56.3 P - 84.1 113 88.7 26.2 1.3 95.6 162.9 105.8 18.4 1
test item 112.5 P - 81.3 94 97.9 17.3 0.8 96.7 108.9 107.7 25.1 1.4
test item 225 P - 82.8 culture was not continued# culture was not continued#
solvent control (acetone) + 100 100 100 13.4 1 100 100 100 3.4 1
positive control (DMBA) 1.1 + 87.4 102.4 83 500.4 37.4 88.3 81.4 106.8 298.6 88.6
test item 7 + 101 147.5 93.1 12.5 0.9 103.5 110.9 110.7 15.5 4.6
test item 14.1 + 101.2 121.9 109 11.2 0.8 93.7 140.7 96.8 7.4 2.2
test item 28.1 + 103.1 115.3 110.7 13.1 1 99.8 105.1 108.8 10.3 3
test item 56.3 P + 102.9 114.1 96.4 6.8 0.5 94.9 103.5 102.2 6.6 2
test item 112.5 P + 100 101.4 119.1 5.8 0.4 91.5 102.5 102.9 5.8 1.7
test item 225 P + 97.8 culture was not continued# 96.3 culture was not continued#
Experiment II / 24h treatment
solvent control (acetone) - 100 100 100 15.2 1 100 100 100 21.6 1
positive control (EMS) 150 - 97.5 104.1 97.4 354.1 23.3 98.3 96.8 86.5 263.2 12.2
test item 7 - 93.2 102 91.1 13.1 0.9 97.1 112 79.2 8.6 0.4
test item 14.1 - 100.8 106.6 100.9 11 0.7 98.2 111.4 86.4 11.2 0.5
test item 28.1 - 98.9 106.1 94.8 23 1.5 95 106.1 92.3 23.5 1.1
test item 56.3 P - 92.8 109.9 102.8 17.6 1.2 87.2 103.9 92.3 8.5 0.4
test item 112.5 P - 94.8 98.8 91.6 12.6 0.8 88.7 115.5 95.2 18.7 0.9
test item 225 P - 88 culture was not continued# 96.1 culture was not continued#
Experiment II / 4h treatment
solvent control (acetone) + 100 100 100 22.9 1 100 100 100 20.3 1
positive control (DMBA) 1.1 + 79.8 72.8 95.4 273.3 11.9 85.3 79.3 89.8 374.8 18.4
test item 7 + 100.7 107 104.2 14.6 0.6 97 89.9 99.2 24.3 1.2
test item 14.1 + 108.5 80.2 102.2 16.8 0.7 98.4 106.2 91.6 16.7 0.8
test item 28.1 + 112.2 82.3 91.9 14.6 0.6 98.7 84.5 100.2 18.2 0.9
test item 56.3 P + 98.4 79.5 103.7 12.3 0.5 100.9 102.2 94.8 22.8 1.1
test item 112.5 P + 103.5 79.7 98.7 16.4 0.7 88.6 127.6 79 14.4 0.7
test item 225 P + 105.2 culture was not continued# 93.1 culture was not continued#

P = Precipitation

# culture was not continued to avoid analysis of too many precipitating concentrations

Conclusions:
In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.
Executive summary:

A mammalian gene mutation assay compliant with GLP and in accordance with OECD guideline 476 was performed to investigate the potential of the test article to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The highest concentration (1800 µg/mL) used in the range finding pre-experiment was limited by the solubility properties of the test item in acetone and aqueous medium. The concentration range of the main experiments was limited by the occurrence of precipitation of the test item. The test item was dissolved in acetone. No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation. Appropriate reference mutagens (EMS and DMBA), used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test substance is considered to be non-mutagenic in this HPRT assay.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Justification for type of information:
see attached justification.
Reason / purpose:
read-across source
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 applicable
Positive controls validity:
valid
Remarks on result:
other: results obtained from read-across
Conclusions:
Based on a gene mutation study performed with the read-across substance, the target substance was not assumed to be mutagenic.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
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:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to
Guideline:
other: Toxicity Test Guideline, (Japan) 1984
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced S-9 liver microsomal fraction
Test concentrations with justification for top dose:
without and with S9 mix:
7 h : 1.0 ; 5.0* ; 10.0* ; 20.0* μg/ml
24 h : 0.1 ; 0.5 ; 1.0 ; 5.0* ; 10.0* ; 20.0* μg/ml
30 h : 1.0 ; 5.0* ; 10.0* ; 20.0* μg/ml
(* precipitation of the test compound in the culture medium during incubation and not evaluated)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen according to its solubility properties and its relative non-toxicity for the cells. The final concentration of the solvent in the culture medium did not exceed 1 % v/v.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without S9 mix Migrated to IUCLID6: final Concentration : 0.72 mg/ml = 5.76 mM
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9 mix Migrated to IUCLID6: final Concentration: 4.20 μg/ml = 15.00 μM
Details on test system and experimental conditions:
PRE-EXPERIMENT FOR TOXICITY: The toxicity of the test compound was determined in a pre-experiment (0.1 - 50 µg/ml) in order to establish a concentration dependent plating efficiency relationship. The experimental conditions in this pre-experiment were the same as described below for the experiment.

DOSE SELECTION
According to the results from this pre-experiment at least six concentrations to be applied in the chromosomal aberration assay were chosen.
The highest dose level used was 10 mM unless limited by the solubility of the test article or that producing some indication of cytotoxicity (reduced plating efficiency and/or partial inhibition of mitosis).
In case of toxic effects the highest dose level should reduce if possible the plating efficiency if possible to approximately 20 - 50 %. In addition, this concentration should suppress if possible mitotic activity (% cells in mitosis) by approximately 50 %, but not so great a reduction that insufficient scorable mitotic cells can be found.
Treatment was performed with the following concentrations:
without and with S9 mix:
7 h : 1.0 ; 5.0* ; 10.0* ; 20.0* μg/ml
24 h : 0.1 ; 0.5 ; 1.0 ; 5.0* ; 10.0* ; 20.0* μg/ml
30 h : 1.0 ; 5.0* ; 10.0* ; 20.0* μg/ml
(* precipitation of the test compound in the culture medium during incubation).
According to the criteria mentioned above, one (7, 30 h) and three concentrations (24 h) were selected to evaluate metaphases for cytogenetic damage.
In the pre-experiment for toxicity the colony forming ability of the CHO cells was only slightly reduced after treatment with 1.0 μg/ml in the presence of S9 mix. However, higher concentrations precipitated in the culture medium during incubation.
In the main experiment, cells after treatment with 1.0 μg/mL as highest dose level could be evaluated for cytogenetic damage. With these concentrations applied in the absence and presence of S9 mix the mitotic index was suppressed.

EXPERIMENTAL PERFORMANCE:
METHOD OF APPLICATION: in medium;
Seeding of the Cultures: Two days old logarithmically growing stock cultures more than 50 % confluent were trypsinised and a single cell suspension
was prepared. The trypsin concentration was 0 .2 % in Ca-Mg-free salt solution.
The cells were seeded into Quadriperm dishes (Heraeus, Hanau, D.) which contained microscopic slides (2 chambers per dish and test group). The medium was DMEM/F12 (1 :1) + 10 % FCS.
Approximately 0.6 - 1.5 x 10 5 cells were seeded into each chamber with regard to fixation interval.
After 48 h the medium was replaced with serum-free medium containing the test compound, either without S9 mix or with 20 μl/ml S9 mix. After 4 h this medium was replaced with normal medium after rinsing twice with "saline G".
All incubations were done at 37° C in a humidified atmosphere with 11.0 % CO2.
5, 21, 27 h after start of the treatment spindle inhibitor is added to the cultures. 2.0 h (7 h interval) or 3.0 h later (24 h and 30 h interval) the cells on the slides in the chambers were treated with hypotonic solution (0.4 % KCl) at 37 °C for 20 min. After incubation in the hypotonic solution the cells were fixed with 3:1 absolute methanol:glacial acetic acid. All two slides per group were prepared. After fixation the cells were stained with giemsa.

NUMBER OF REPLICATIONS: 2

SPINDLE INHIBITOR (cytogenetic assays): colcimid (0.2 µg/ml culture medium).

ANALYSIS
- Analysis of Metaphase Cells: The slides were evaluated using NIKON microscopes with 100x oil immersion objectives. Gaps, breaks, fragments, deletions, exchanges and chromosomal disintegrations are recorded as structural chromosome aberrations.

NUMBER OF CELLS EVALUATED: At least 100 well spread metaphases per slide (200 per test group) were scored for cytogenetic damage on coded slides. Only metaphases with characteristic chromosome number of 20 ± 1 are included in the analysis.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (% cells in mitosis)
Evaluation criteria:
A test substance is classified as mutagenic if it induces either a significant dose-related increase in the number of structural chromosomal aberrations or a significant positive response for at least one of the test points.
A test article producing neither a significant dose-related increase in the number of structural chromosomal aberrations nor a significant positive response at any one of the test points is considered non-mutagenic in this system.
Statistics:
A statistical evaluation of the results was not necessary to perform. The aberration rates of the test groups after treatment with the test article were in the range of the control values.
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:
valid
Positive controls validity:
valid
Additional information on results:
In the pre-experiment on toxicity (colony forming ability) in the presence of S9 mix after treatment with 1.0 μg/ml the colony forming ability was only slightly reduced. However, higher concentrations precipitated in the culture medium during incubation.
In the main experiment, the mitotic index was reduced after treatment with 1.0 μg/ml indicating that the substance had cytotoxic properties.

Summarv of results

Fixation Interval: 7 h % aberrant cells
article number of cells analyzed concentration /ml S9-Mix incl. Gaps excl. Gaps exchanges
solvent control 200 0 - 0.5 0 0
test article 200 1 - 3.5 2.5 0.5
solvent control 100* 0 + 7 4 1
test article 100* 1 + 8 4 0
Fixation Interval: 24 h % aberrant cells
article number of cells analyzed concentration /ml S9-Mix incl. Gaps excl. Gaps exchanges
negative control 200 0 - 2.5 2.5 1
solvent control 200 0 - 0.5 0 0
positive control EMS 200 0.72 - 26.5 23.5 12.5
test article 200 0.1 - 1.5 1 0.5
test article 200 0.5 - 4 2.5 0.5
test article 200 1 - 4 3 0
negative control 200 0 + 4.5 4 1.5
solvent control 100* 0 + 9 5 2
positive control CPA 200 4.2 + 15 10.5 8
test article 200 0.1 + 9.5 5 1.5
test article 200 0.5 + 5 2.5 0.5
test article 200 1 + 7 2 0
1
Fixation Interval: 24 h % aberrant cells
article number of cells analyzed concentration /ml S9-Mix incl. Gaps excl. Gaps exchanges
solvent control 200 0 - 4.5 1 0
test article 200 1 - 4.5 2.5 0.5
solvent control 200 0 + 4 3.5 1
test article 200 1 + 7.5 4 1.5

*one slide out of two was not scorable

Conclusions:
In the study described and under the experimental conditions reported, the test article did not induce structural chromosome aberrations as determined by the chromosomal aberration test in the CHO Chinese Hamster cell line and is therefore considered to be non-mutagenic in this chromosomal aberration test.
Executive summary:

The test article was assessed for its potential to induce structural chromosome aberrations in CHO cells in vitro. Preparation of chromosomes was done 7 h (high dose), 24 h (low, medium and high dose) and 30 h (high dose) after start of treatment with the test article. The treatment interval was 4 h. In each experimental group two parallel cultures were used. Per culture 100 metaphases were scored for structural chromosomal aberrations. The following dose levels were evaluated with and without S9 mix: 7 h: 1.0 μg/ml; 24 h: 0.1; 0.5; 1.0 μg/ml; 30 h: 1.0 μg/ml. The concentration range of the test article applied had been determined in a pre-experiment using the plating efficiency assay as indicator for toxicity response. Treatment of the cells with 1.0 µg/ml reduced only slightly the plating efficiency. Higher concentrations than 1.0 µg/ml precipitated in the culture medium during incubation. However, the mitotic index was reduced with the highest concentration in the absence and presence of S9 mix. There was no relevant increase in cells with structural aberrations after treatment with the test article at any fixation interval either without or with metabolic activation by S9 mix. Appropriate reference mutagens were used as positive controls and showed distinct increases of cells with structural chromosome aberrations .

Therefore, the test article is considered to be non-mutagenic in this chromosomal aberration test.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Justification for type of information:
see attached justification
Reason / purpose:
read-across source
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:
valid
Positive controls validity:
valid
Remarks on result:
other: results obtained from read-across
Conclusions:
Based on results obtained from a chromosome aberration study performed with the read-across substance, the target substance was not assumed to be clastogenic.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Additional information from genetic toxicity in vitro:

Reliable data from mutagenicity studies are available for the test article and for other members of the "Perylene based pigments" category (see attached category justification). All of these data are taken into account for the evaluation and assessment of the acute toxicity of the test article.

Bacterial Mutagenicity

Several Ames tests are available addressing the test article's potential to cause point mutations in bacteria. The majority of studies were clearly negative, some reports, however, had equivocal results. In the following all available data is assessed in order to derive a final conclusion regarding bacterial mutagenicity.

In the most recent study performed according to OECD guideline 471 and in compliance with GLP, the test substance was tested for its mutagenic potential in a reverse mutation assay (Ames standard plate test and Prival preincubation test) using Salmonella typhimurium strains TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA. The Prival preincubation test is a modification of the standard Ames reverse mutation assay, in which flavin mononucleotide (FMN), liver S9 mix from uninduced hamsters and a preincubation step are used to facilitate azo reduction and the detection of the resulting mutagenic aromatic amines. This test is therefore the most appropriate method for the investigation of azo-dyes and diazo compounds such as the test compound. Both assays were performed in the presence and absence of a metabolic activation system at concentrations ranging from 33 to 5000 µg/plate. Precipitation of the test substance was found from 333 μg/plate onward with and without S9 mix. A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions from 2500 μg/plate onward. According to the results of the present study, the test substance did not lead to a biologically relevant increase in the number of revertant colonies either with or without S9 mix in two experiments carried out independently of each other. The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain. In addition, the positive control substances both with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data or above. Thus, under the experimental conditions of this study, the test substance is not mutagenic in the Ames standard plate test and in the Prival preincubation test in the absence and the presence of metabolic activation. Based on the appropriate modifications to the protocol while utilizing most recent test material, this study was designated as the leading key study (BASF, 2011).

This result is supported by the data obtained in another GLP-compliant Ames test performed with test substance of very high purity (close to 100%) following OECD guideline 471. The study was performed with Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2 uvr A at 20 to 5000 µg/plate in the standard plate test and at 4 to 2500 µg/plate in the Prival preincubation test with and without metabolic activation. Similar to the report from 2011, the Prival modification was chosen because the substance is an azo-dye. An increase in the number of his+ or trp+ revertants was not observed either in the standard plate test or in the Prival preincubation test with or without S9-mix. A slight decrease in the number of revertants was observed only using the Salmonella strains in the standard plate test depending on the strain and test conditions from about 2500 µg/plate onward. Precipitation of the test substance was found from about 4 µg/plate onward. Under the conditions tested, the test substance was considered to be not mutagenic (BASF, 2002).

In another study the test substance was investigated in a GLP-compliant Ames test according to OECD guideline 471 using Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2 uvr A at 20 to 5000 µg/plate (TA98: 20 – 6000 µg/plate) in the standard plate test with and without metabolic activation. A slight decrease in the number of revertants was occasionally observed depending on the strain and test conditions from about 2500 µg/plate onward. Test substance precipitation was found from about 20 µg/plate onward. According to the results of the present study, the test substance led to a slight increase in the number of his+ revertants with the strain TA98 in two experiments carried out independently of each other with S-9 mix (from about 3,000 µg/plate (factor 2.2) onward with an increase in the number of revertant colonies by a factor of 3.0 at 6,000 µg/plate). However, the weakly positive reaction was observed at high doses only at which evident test substance precipitation was found and thus, impurities rather than the test substances itself might be the cause for the positive result. No increase in the number of his+ or trp+ revertants were observed for all other strains. In conclusion, under the conditions of this study, the test article was found weakly mutagenic in S. typhimurium TA98 in the presence of S9-mix, which might be caused by impurities rather than the test substance itself (BASF, 1999).

A similar slightly positive result of Salmonella typhimurium strain TA98 was also obtained in an Ames test performed by the Litron Laboratories in 1984. In this study, the test article was tested using S. typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 at 1 - 5000 µg/plate with and without metabolic activation in a standard plate test. The test substance was found weakly mutagenic only using the two frameshift strains TA1538 and TA98 and only after the addition of a metabolizing system. However, the slight increase in the number of his revertants was merely observed at the highest dose of 5000 µg/plate at which it was very probable that the dyestuff was no longer soluble and that test substance precipitation occurred (this assumption is based on the fact that below 1000 µg/plate the colonies were scored automatically using a colony counter but from about 1000 µg/plate onward the samples were hand counted, however, without giving any explanation for this). Furthermore, it should be expected that at doses of 5000 µg/plate the capacity of the S-9 mix might be overloaded for metabolizing such an amount of test substance. Thus, it cannot be ruled out that impurities rather than the compound itself might be the cause for the weakly positive reaction (Litron Laboratories Ltd., 1984).

In a supporting follow up Ames study the test article was tested at concentrations of 20 - 7500 µg/plate with and without metabolic activation in a standard plate test. The aim of the study was to refute or to confirm the findings from the above study by Litron Labs using a test item batch with a purity of 99% and only using the frameshift strain TA 98. According to the results of this study, the test substance is again weakly mutagenic under the experimental conditions chosen. The increase in the number of his revertants was approximately in the same order of magnitude compared to the previous study, and was observed under the same conditions, i.e. only with S-9 mix and only at high dose levels, where the solubility already was by far exceeded. Therefore, also in the case of this study impurities cannot be ruled out as the cause for the slight increase in the number of mutant colonies (BASF AG, 1987).

In another follow up study, six different production charges of the test item were tested in an Ames test performed with S. typhimurium strain TA 98 at concentrations of 20 – 5000 µg/plate in the presence or absence of S9-mix (only summary available). Two of the tested batches were clearly negative. The other four batches, however, showed slight increases in the number of revertants, but again only at high concentrations (2500 to 5000 µg/plate), where precipitation occurred. The test article was reported precipitating from concentrations of 100 µg/plate and higher (BASF, 1990)

In two additional Ames tests only available as summaries, two different batches of the test article were tested in Salmonella typhimurium strains TA 100 and TA 98 at concentrations of 100 – 5000 µg/plate with or without S9 mix. In this study, all results were negative, either in the presence or absence of a metabolic activating system up to and including the highest concentrations tested. The results of these studies clearly demonstrate that the weak positive response observed some studies are batch dependent and therefore impurities are most likely the cause for the positive results rather than the test article itself. (BASF, 1991)

Taking all data together, it can be concluded that some batches of the test article caused weak mutagenic reactions in S. typhimurium strain TA 98 (and TA 1538), however only at high concentrations where precipitation of the test article occurred. It is therefore very likely that impurities rather than the substance itself are the cause for the weak positive reactions observed infrequently. Since the last two tests performed in 2002 and 2011 were clearly negative up to precipitating concentrations, the assumption can be made that recent production charges of this compound no longer include the impurity that might have caused the weak positive responses in past tests. It is therefore concluded that the test substance does not raise any concern regarding bacterial mutagenicity and is considered as not mutagenic.

In addition, further Ames tests are available for all other category members (at least one per substance). None of these tests gave any rise to concern for genotoxicity. Consequently, all substances of this category have been regarded as not genotoxic in the bacterial reverse mutation test.

Mammalian Mutagenicity

No data for the test substance is available regarding mutagenicity in mammalian cells. However, valid HPRT assays are available for two other category members.

A GLP-compliant HPRT assay is available for the read-across substance (BASF, 2012). The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The highest concentration (1800 µg/mL) used in the range finding pre-experiment was limited by the solubility properties of the test item in acetone and aqueous medium. Concentrations chosen both for cells treated with and without metabolic activation in the main experiment were 7.0, 14.1, 28.1, 56.3, 112.5, and 225.0 µg/ml.

The concentration range of the main experiments was limited by the occurrence of precipitation of the test item. No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation. Therefore, under the experimental conditions reported, the test substance is considered to be non-mutagenic in this HPRT assay.

In another HPRT test according to OECD guideline 476 and in compliance with GLP, another category member was investigated for its mutagenic potential to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster (BASF, 2012). The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. Concentrations chosen were 5.6, 10.8, 21.5, 43.0, 86.0, 172.0 µg/ml and 10.8, 21.5, 43.0, 86.0, 172.0, 344.0 µg/ml for cells treated with and without metabolic activation, respectively.

The concentration range of the main experiments was limited by the occurrence of precipitation of the test item. No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation. Therefore, under the experimental conditions reported, the test substance is considered to be non-mutagenic in this HPRT assay.

Chromosomal Damage

No data for the test substance is available regarding clastogenicity. However, valid in vitro chromosomal aberration tests are available for three other category members. The test articles were tested in the presence and absence of a metabolic activating system. None of these tests gave rise to concern for genotoxicity up to and including the highest dose tested. Consequently, all three substances have been regarded as not clastogenic. According to the category approach the data available for other category members is used to assess the toxicity of the test article. Based on the available and reliable data available for three category members, the test substance is not considered to have the potential to cause chromosome damage.

For the read-across substance chosen here, chromosome aberration assay was performed according to OECD 473 in compliance with GLP (BASF, 1989). CHO cells were treated with concentrations ranging from 0.1 to 20.0 µg/ml both with and without metabolic activation. Exposure period was 4 h in three independent experiments. Different fixation intervals were chosen for the experiments, namely 7h, 24 h, and 30 h. All three experiments did not give evidence for a clastogenic potential of the test substance. The mitotic index was reduced after treatment with 1.0 µg/ml indicating that the test substance had cytotoxic properties.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for the purpose of classification under Regulation (EC) No.1272/2008. Based on the data, classification for genotoxicity is not warranted under Regulation (EC) No.1272/2008.