Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance was not mutagenic in two Ames tests. No potential to cause mutations in mammalian cells or damage to chromosomes is expected based on data obtained from other category members.

Link to relevant study records

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Endpoint:
in vitro gene mutation study in bacteria
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 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Jul 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
his and 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
Test concentrations with justification for top dose:
20 - 5000 µg/plate (standard plate test) and 4 - 1000 µg/plate (preincubation test)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the limited solubility/insolubility of the test substance in water, DMSO was selected as the 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: 2-aminoanthracene
Remarks:
with S9 mix; all strains
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
Remarks:
without S9 mix; TA 1535 and TA 100
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylenediamine
Remarks:
without S9 mix; TA 98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9 mix; TA 1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
without S9 mix; E. coli WP2 uvrA
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
Plate incorporation method:
- Exposure duration: ca. 48-72 hours at 37°C
Preincubation method:
- Preincubation period: 20 minutes at 37°C
- Exposure duration: ca. 48-72 hours at 37°C

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertants, clearing or diminution of the background lawn (= reduced his- or trp- background growth), reduction in the titer
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 S-9 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 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:
- Toxicity: A slight decrease in the number of revertants was occasionally observed.
- Precipitation: Precipitation of the test substance was found from about 100 μg/plate onward.

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 24 37 113 114 19 20 9 10 33 40
20 23 33 101 113 20 20 9 8 27 37
100 23 28 73 107 20 21 9 9 27 35
500 22 26 104 110 19 21 8 9 23 30
2500 22 25 105 108 20 17 7 9 27 31
5000 22 24 98 106 20 19 6 7 31 27
MNNG 1145 1020
2-AA 1327 1058 219 103 291
AAC 777
NOPD 1164
ENNG 873

Results of Experiment II (Pre-incubation 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 28 36 103 112 18 18 10 11 28 35
4 20 29 101 110 14 15 11 11 28 29
20 19 28 105 121 14 15 10 11 28 32
100 18 22 103 101 11 15 9 10 31 31
500 16 24 95 101 11 12 8 10 28 31
1000 14 21 89 104 9 11 9 11 30 33
MNNG 931 1017
2-AA 959 1054 151 176 195
AAC 676
NOPD 675
ENNG 843

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)

ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine (10 µg/plate)

Conclusions:
According to the results of the present study, the test substance is not mutagenic in the Salmonella typhimurium/ Escherichia coli reverse mutation assay under the experimental conditions chosen here.
Executive summary:

According to the results of the present study, the test substance did not lead to an increase in the number of revertant colonies either without S-9 mix or after adding a metabolizing system in two experiments carried out independently of each other (standard plate test and preincubation assay.

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 test 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 a chromosome aberration test conducted with the read-across substance, the target substance was not assumed to be clastogenic.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
his and trp
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100, TA 1538 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver S-9 mix
Test concentrations with justification for top dose:
4 - 3500 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with S9 mix; all strains
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9 mix; TA 1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Methylhydrazone Derivative
Remarks:
without S9 mix; TA 98, 100 and 1538
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Streptocotocine
Remarks:
without S9 mix; TA 1535
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ENNG
Remarks:
without S9 mix; E.coli WP2 uvrA
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: ca. 48 hours at 37°C

NUMBER OF REPLICATIONS: 4
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100, TA 1538 and E. coli WP2
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
Additional information on results:
- Precipitation: Precipitation of the test substance was found from about 500 μg/plate onward.
Conclusions:
In the study presented, the test substance was investigated for its genotoxic potential in the reverse bacteria mutation assay. The test substance was found to be non-mutagenic under the conditions chosen.
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

The test article was tested in the Ames reverse mutation assay (GLP-compliant study 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 in the standard plate test and at 4 to 1000 µg/plate in the preincubation test (vehicle: DMSO) with and without metabolic activation (BASF AG, 1998). An increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S-9mix or after the addition of a metabolising system.A slight decrease in the number of revertants was occasionally observed. Precipitation of the test substance was found from about 100 μg/plate onward. Under the conditions tested, the test substance is not mutagenic in the Ames test.

In a second Ames reverse mutation assay (study equivalent to OECD guideline 471) the test substance was tested using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 and Escherichia coli WP2 uvr A at 4 to 3500 µg/plate (vehicle: DMSO) with and without metabolic activation using the plate incorporation test (Hoechst AG, 1982). Precipitation of the test substance was found from about 500 μg/plate onward. Under the conditions tested, the test article was not mutagenic in the Ames test.

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 genotoxicty. 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.

The read-across substance was tested in a chromosomal aberration assay according to OECD 473 and in compliance with GLP (BASF, 1989). Chinese hamster ovary cells (CHO) were treated both with and without metabolic activation to concentrations ranging from 0.1 to 20.0 µg/ml. Exposure period was 4 h in three independent experiments. Different fixation intervals were chosen for the experiments, namely 7 h, 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 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.