Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance did not cause gene mutations in Salmonella typhimurium and Escherichia coli (Ames test). The substance was also negative in a chromosome aberration test. No potential to cause mutations 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
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:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
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, 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 1254 induced rat liver S-9 mix
Test concentrations with justification for top dose:
20, 100, 500, 2500, 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
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: TA100 and TA1535
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylendiamine
Remarks:
without S9 mix: TA98
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: TA1537
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
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
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
Plate incorporation method:
- Exposure duration: ca. 48 hours at 37°C
Preincubation method:
- Preincubation period: 30 minutes at 30°C
- Exposure duration: 48 hours at 37°C

NUMBER OF REPLICATIONS:
- 3 test plates per dose or per control

DETERMINATION OF CYTOTOXICITY
reduced his- or trp- background growth
Evaluation criteria:
In general, a substance to be characterized as positive in the bacterial tests has to fulfill the following requirements:
- doubling of the spontaneous mutation rate ( control)
- dose-response relationship
- reproducibility of the results
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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: from about 2500 µg/plate onward

Standard Plate Test Result:

TA 1535 TA 100 TA 1537 TA 98 WP2uvrA
Dose (µg/plate) -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
0 10 12 88 102 10 10 27 35 36 47
20 10 13 92 115 11 12 22 35 26 39
100 9 13 74 98 9 10 31 33 29 38
500 15 8 67 94 9 10 29 40 33 37
2500 14 12 90 102 9 9 26 38 29 34
5000 12 16 77 92 9 12 26 41 36 34
2-AA 320 908 121 2350 173
MNNG 1336 1327
AAC 463
NPD 1817
ENNG 199

Preincubation Test Result:

TA 1535 TA 100 TA 1537 TA 98 WP2uvrA
Dose (µg/plate) -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
0 17 18 116 113 11 11 29 35 22 24
20 17 15 123 120 9 10 24 39 25 28
100 16 18 97 119 9 10 27 42 26 27
500 20 18 79 107 10 12 28 40 23 30
2500 14 17 85 89 10 10 21 40 25 30
5000 16 15 69 81 12 10 22 37 22 24
2-AA 151 661 109 1137 127
MNNG 879 897
AAC 230
NPD 1067
ENNG 399

Controls:

2-AA: 2-aminoanthracene (10 µg/plate for TA strains, 60 µg/plate for WP2uvrA)

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

AAC: 9-aminoacridine (100 µg/plate)

NPD: 4-nitro-o-phenylendiamine (10 µg/plate)

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

Conclusions:
According to the results of the present study, the test substance is not mutagenic in the Ames test and in the Escherichia coli reverse mutation assay under the experimental conditions chosen here.
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 gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 05, 2011 - December 09, 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 supplemented with 10 % fetal bovine serum (FBS), neomycin (5 Pg/mL) and amphotericin B (1 %). The cell cultures were incubated at 37 °C in a 1.5 % carbon dioxide atmosphere (98.5 % air).
- 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: 10.8; 21.5; 43.0; 86.0; 172.0; 344.0 µg/ml
With S9 mix: 5.6; 10.8; 21.5; 43.0; 86.0; 172.0 µg/ml
The cultures at the lowest concentrations with metabolic activation were not continued, since a minimum of only four analysable concentrations is required by the guidelines. The cultures at the maximum concentration without metabolic activation were not continued to avoid evaluation of too many precipitating concentrations.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative nontoxicity to the cell cultures.
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). Test item concentrations between 43.0 and 5500 µg/mL were used to evaluate cytotoxicity in the presence (4 hours treatment) and absence (4 hours and 24 hours treatment) of metabolic activation.
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 171.9 µg/mL and above without metabolic activation following 4 and 24 hours treatment and at 85.9 µg/mL and above with metabolic activation (4 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:
cytotoxicity
Remarks:
Toxic effects only observed in the pre-test at high concentrations
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 86.0 µg/mL and above with metabolic activation (4 hours treatment) and without metabolic activation (4 and 24 hours treatment).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant toxic effects occurred at the maximum concentration of 5500 µg/mL without metabolic activation following 4 hours treatment. In the presence of metabolic activation (4 hours treatment) no relevant cytotoxicity were observed up to the maximum concentration. Following 24 hours treatment without metabolic activation, increasing cytotoxicity was observed at 343.8 µg/mL and above.

An increase of the induction factor exceeding the threshold of three times the mutation frequency of the corresponding solvent control was observed in the first culture of the second experiment with metabolic activation at 86.0 µg/mL. However, the increase was based on a rather low mutation frequency of the solvent control of just 6.1 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 (water) - 100 100 100 8.6 1 100 100 100 23.8 1
positive control (EMS) 150 - 90.4 93.1 78.1 171.2 19.8 80 106.3 76.5 123.1 5.2
test item 10.8 - 96.5 139.9 92 12.7 1.5 96 112.9 89.4 16 0.7
test item 21.5 - 106.4 123.7 89.9 10.2 1.2 97.6 108.1 77.8 20.3 0.9
test item 43 - 98 100.3 69.9 13.8 1.6 99.1 68.1 90.5 11.6 0.5
test item 86 P - 93.1 112.6 68.7 21 2.4 101.6 82.9 66.9 11.5 0.5
test item 172 P - 79.8 82.6 87.5 9.8 1.1 101.6 95 68.1 18.7 0.8
test item 344 P - 77.7 culture was not continued# 91.9 culture was not continued#
solvent control (water) + 100 100 100 8.5 1 100 100 100 9.5 1
positive control (DMBA) 1.1 + 44 38.7 69.8 754.4 88.3 46.9 49.6 67.8 856.7 90.4
test item 5.6 + 91.1 culture was not continued## 98.2 culture was not continued##
test item 10.8 + 86.3 80.3 91.8 9 1.1 108.4 103.2 94.1 6.1 0.6
test item 21.5 + 85.6 88.3 119 7.4 0.9 102.2 88.3 90.3 4.8 0.5
test item 43 + 94.5 90.4 103.3 7.8 0.9 100.4 85.9 99.5 15.1 1.6
test item 86 P + 88.3 103.1 112.5 20.9 2.4 102.9 107.2 86.9 10.1 1.1
test item 172 P + 84 81.9 99.9 24.4 2.9 99.2 107.7 92.5 25.6 2.7
Experiment II / 24h treatment culture I culture II
solvent control (water) - 100 100 100 16.3 1 100 100 100 10.8 1
positive control (EMS) 150 - 83.6 60.8 107.2 410.7 25.2 98 108.7 99.6 307.2 28.5
test item 10.8 - 97.1 79.5 105.4 16 1 96.6 119.3 114.5 14.3 1.3
test item 21.5 - 91.4 83.8 102.7 18 1.1 97.4 110.7 110.2 14.1 1.3
test item 43 - 87.5 66.7 101.7 27.5 1.7 95.6 130.8 104.3 13.4 1.2
test item 86 P - 78.9 80.1 105 22.5 1.4 84.3 112 104.3 22.9 2.1
test item 172 P - 71.8 69.1 106.2 18.3 1.1 70.8 109.7 100.4 10.3 1
test item 344 P - 50.4 culture was not continued# 51.1 culture was not continued#
Experiment II / 4h treatment
solvent control (water) + 100 100 100 6.1 1 100 100 100 11 1
positive control (DMBA) 1.1 + 44 57.1 93 588.3 96 53.7 74.3 91.9 635.3 58
test item 5.6 + 111.7 culture was not continued## 100.4 culture was not continued##
test item 10.8 + 100.9 79.5 104.5 9.6 1.6 88.2 98.3 96.4 11.4 1
test item 21.5 + 110.3 105.4 99 7.7 1.3 92.7 90 92.1 15.4 1.4
test item 43 + 106.8 111.9 102.4 9.2 1.5 93 114.8 104.4 13.9 1.3
test item 86 P + 98.6 79.6 73.5 23.7 3.9 89.7 126.6 98.6 14.8 1.4
test item 172 P + 109 81.2 94.4 8.2 1.3 89 101.5 93.8 11.5 1

P = Precipitation

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

## culture was not continued as a minimum of only four analysable concentrations is required

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 (5500 µg/mL) used in the range finding pre-experiment was chosen with respect to the current OECD guideline 476 and the purity of the test item. The test item was dissolved in deionised water. 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. 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:
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 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 item was tested in the Ames reverse mutation assay (according to OECD guidelines 471 and 472) using Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2 uvrA at 20 to 5000 µg/plate with and without metabolic activation using DMSO as the vehicle (BASF AG, 1991). In the toxicity test, the test compound proved to be not toxic to the bacteria up to 5000 µg/plate. Incomplete solubility of the test substance in DMSO was observed from about 2500 µg/plate onward.In the absence of the metabolic activation system the test compound did not show a dose dependent increase in the number of revertants in any of the bacterial strains. Also in the presence of the metabolic activation system, treatment of the cells with test material did not resultant in relevant increases in the number of revertant colonies. Under the conditions tested, the test article is not mutagenic in bacteria.

In a second Ames reverse mutation assay (study comparable to OECD guideline 471) the test article was tested using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 at 1 to 5000 µg/plate (vehicle: DMSO) with and without metabolic activation (BASF AG, 1985). The test material was not mutagenic in any of the S. typhimurium strains. Under the conditions tested, the test article is 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 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 in HPRT assays are available for two other category members.

In an HPRT test according to OECD guideline 476 and in compliance with GLP, the read across test substance 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.

An additional GLP-compliant HPRT assay is available for another category member (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.

Chromosomal Damage

The test article was studied in a GLP-compliant in vitro mammalian chromosome aberration test with Chinese hamster ovary cells (CHO) following OECD guideline 473 (CCR, 1989). The treatment interval was 4 hours. Preparation of chromosomes was done 7 hours (1.0 µg/L), 24 hours (0.1, 0.5 and 1.0 µg/L) and 30 hours (1.0 µg/L) after start of treatment with the test article in absence and presence of S9-mix. 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. 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. Therefore, the substance is considered to be non-mutagenic in this chromosomal aberration test.

This finding is supported by the results obtained in two additional chromosomal aberration tests performed with two 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, both substances have been regarded as not clastogenic.

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.