7,14,25,32-tetraazaundecacyclo[21.13.2.2²,⁵.0³,¹⁹.0⁴,¹⁶.0⁶,¹⁴.0⁸,¹³.0²⁰,³⁷.0²⁴,³².0²⁶,³¹.0³⁴,³⁸]tetraconta-1(36),2(40),3,5(39),6,8(13),9,11,16,18,20,22,24,26(31),27,29,34,37-octadecaene-15,33-dione; 7,14,25,32-tetraazaundecacyclo[21.13.2.2²,⁵.0³,¹⁹.0⁴,¹⁶.0⁶,¹⁴.0⁸,¹³.0²⁰,³⁷.0²⁵,³³.0²⁶,³¹.0³⁴,³⁸]tetraconta-1(36),2(40),3,5(39),6,8(13),9,11,16,18,20,22,26(31),27,29,32,34,37-octadecaene-15,24-dione

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames-Test: negative, according to OECD TG 471, GLP compliant, Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli WP2 uvrA, with and without metabolic activation, 2006, K1

 

HPRT: negative, according to OECD TG 476, GLP compliant, Chinese hamster lung fibroblasts (V79), with and without metabolic activation, negative, 2012, K1

 

Chromosome aberration test: negative, according to OECD TG 473, GLP compliant, Chinese hamster lung fibroblasts (V79), with and without metabolic activation, negative, 2006, K1

Link to relevant study records

Reference

Reference 1

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

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

GLP compliance:

yes (incl. QA statement)

Remarks:

Harlan Cytotest Cell Research GmbH

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

- Physical state: Solid, black powder
- Analytical purity: > 99%
- Storage condition of test material: Room temperature

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 / 10^6cells	induction factor	relative cloning efficiency I (%)	relative cell density (%)	relative cloning efficiency II (%)	mutant colonies / 10^6cells	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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 Oct 2005 - 13 Apr 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

(1997)

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

- Physical state: Solid, black powder
- Analytical purity: > 99%
- Storage condition of test material: Room temperature

Target gene:

not applicable

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM medium with glutamine supplemented with − 10% (v/v) fetal calf serum (FCS),
− 1% (v/v) penicillin/streptomycin (10 000 IU / 10 000 μg/mL)
− 1% (v/v) amphotericin B (250 μg/mL)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically checked for plating efficiency (= colony forming ability) incl. vital staining: yes

Metabolic activation:

with and without

Metabolic activation system:

The S-9 fraction was prepared according to AMES et al. (Mut. Res. 31, 347-364, 1975) from the livers of at least 5 male Sprague-Dawley rats that had received a single ip injection of 500 mg Aroclor 1254/ kg bw 5 days earlier.

Test concentrations with justification for top dose:

first experiment:
4-hour exposure, 18-hour sampling time, without S-9 mix: 0; 1.56; 3.13; 6.25; 12.5; 25.0; 50.0; 75.0; 100.0 μg/mL
4-hour exposure, 18-hour sampling time, with S-9 mix: 0; 1.56; 3.13; 6.25; 12.5; 25.0; 50.0; 75.0; 100.0 μg/mL

Dose selection was based on the solubility of the test substance, i.e. doses > 12.5 μg/mL both with and without S-9 mix led to strong precipitation which interferes with evaluation of metaphases.

second experiment:
18-hour exposure, 18-hour sampling time, without S-9 mix: 0; 0.78; 1.56; 3.13; 6.25; 12.5; 25.0 μg/mL
18-hour exposure, 28-hour sampling time, without S-9 mix: 0; 3.13; 6.25; 12.5 μg/mL
4-hour exposure, 28-hour sampling time, with S-9 mix: 0; 1.56; 3.13; 6.25; 12.5; 25.0 μg/mL

Again doses ≥ 12.5 μg/mL led to strong test substance precipitation which interferes with the evaluation of metaphases.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO was considered the most suitable. Therefore, DMSO was selected as the vehicle, which had been demonstrated to be suitable in the V79 in vitro cytogenetic test and for which historical control data are available.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without S-9: 500 µg/mL ethyl methanesulfonate (EMS); With S-9: 500 µg/mL cyclophosphamide (CPP)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium in Quadriperm dishes

DURATION
- Preincubation period: 24 - 30 hrs
- Exposure duration: 4 or 18 hrs

- Expression time (cells in growth medium): 14 hrs or 24 hrs; after continuous treatment, i.e. 18 hours without S-9 mix, cells were treated in culture medium supplemented with 10% FCS and in the case of a sampling time of 28 hrs incubated again for another 10 hours.

- Fixation time (start of exposure up to fixation or harvest of cells): 2 - 3 hours prior to harvesting the cells, 0.2 μg Colcemid/mL culture medium was added to each chamber in order to arrest mitosis in the metaphase. For hypotonic treatment, 5 mL of a 0.4% KCl solution which was at 37°C was added for about 20 min. Subsequently, 5 mL of fixative (methanol : glacial acetic acid/3 : 1) which was added at 4°C and kept for at least 15 min and then replaced. After about another 10 min, the fixative was replaced again and kept for at least 5 min at room temperature for complete fixation.

SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): solution of Giemsa and Titrisol (15 mL Giemsa, 185 mL Titrisol pH 7.2) for 10 minutes

NUMBER OF REPLICATIONS: 2 chambers of Quadriperm dishes were used per test culture.

NUMBER OF CELLS EVALUATED: 200 metaphases evaluated

CELL MORPHOLOGY
About 3 hours and 16 - 18 hours after test substance treatment, cultures of all test groups were checked for cell morphology, which is an indication of attachment of the cells to the slides.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
A mitotic index based on 1 000 cells/culture was determined for all evaluated test groups in both experiments.
For the determination of cytotoxicity, additional cell cultures (using 25 cm2 plastic flasks) were treated in the same way as in the main experiment. Growth inhibition was estimated by counting the number of cells in the dose groups in comparison with the concurrent vehicle control at the end of the culture period using a counter.

CELLCYCLE
The cell cycle of the untreated V79 cells lasted for about 13 - 14 hours under the selected culture conditions. Thus, the selected 1st sampling time of 18 hours was within the 1 - 1.5 x the normal cell cycle time, as recommended by the OECD TG 473. The later sampling time of 28 hours was chosen to cover a possible cell cycle delay.

Evaluation criteria:

If there is a clear increase in chromosomally damaged cells, the number of metaphases to be analyzed is reduced from the planned 200 mitoses/test group.
For details concerning evaluation criteria, please refer to "Any other information on materials and methods incl. tables".

Assessment criteria
The test chemical was assessed as “positive” in this assay if the following criteria were met:
• A dose-related and reproducible significant increase in the number of cells with structural / numerical chromosomal aberrations.
• The number of aberrant cells exceeded both the concurrent negative control range and the highest value of the negative historical control range.

A test substance generally was considered as “negative” if the following criteria were met:
• The number of cells with structural / numerical aberrations in the dose groups was not significantly above the concurrent negative control and was within the historical control data.

Statistics:

The proportion of metaphases with aberrations was calculated for each group.
A comparison of each dose group with the vehicle control group was carried out using Fisher's exact test for the hypothesis of equal proportions. This test was Bonferroni-Holm corrected versus the dose groups separately for each time and was performed one-sided. If the results of this test were significant, labels (* p < 0.05, ** p < 0.01) are printed in the tables.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At concentrations of 25 µg/mL and above, evaluation was not possible due to strong test substance precipitation which interferes with metaphase evaluation.

Mitotic index:
According to the results of the determination of the mitotic index, no suppression of the mitotic activity was observed under any of the experimental conditions.

Cell counts:
According to the results of the cell count, no growth inhibition was observed under any of the experimental conditions.

Cell morphology:
Cell attachment was not influenced at any dose evaluated for structural chromosomal aberrations.

CHROMOSOME ANALYSIS - 1ST EXPERIMENT

 

- Clastogenic mode of action: After a treatment time of 4 hours no increase in the number of chromosomally damaged cells was observed either without S-9 mix or after the addition of a metabolizing system.

- Aneugenic mode of action: No increase in the number of cells with changes in the number of chromosomes was demonstrated either without S-9 mix or after the addition of a metabolizing system.

 

 

CHROMOSOME ANALYSIS - 2ND EXPERIMENT

 

- Clastogenic mode of action: Both with and without S-9 mix, no increase in the number of structurally damaged metaphases was observed either after a treatment time of 4 hours or after a continuous treatment of 18 hours at both sampling times, i.e. 18 hours and 28 hours.

- Aneugenic mode of action: No increase in the number of cells with changes in the number of chromosomes was demonstrated either without S-9 mix or after the addition of a metabolizing system.

 

SUMMARY TABLE

					Metaphases with Aberrations
					Incl Gaps		Exc. Gaps
Exposure Time	Sampling Time	Dose (µg/ml)	S9-Mix	number of metaphases	n	%	n	%
4 h	18 h	Vehicle	-	200	11	5.5	4	2
4 h	18 h	3.13	-	200	10	5	5	2.5
4 h	18 h	6.25	-	200	7	3.5	2	1
4 h	18 h	12.5	-	200	5	2.5	4	2
4 h	18 h	EMS 500	-	100	19	19	15	15
4 h	18 h	Vehicle	+	200	9	4.5	3	1.5
4 h	18 h	3.13	+	200	12	6	7	3.5
4 h	18 h	6.25	+	200	15	7.5	4	2
4 h	18 h	12.5	+	200	8	4	3	1.5
4 h	18 h	CPP 0.5	+	100	31	31	26	26
18 h	18 h	Vehicle	-	200	6	3	2	1
18 h	18 h	3.13	-	200	13	6.5	7	3.5
18 h	18 h	6.25	-	200	6	3	2	1
18 h	18 h	12.5	-	200	4	2	0	0
18 h	18 h	EMS 500	-	100	19	19	19	19
18 h	28 h	Vehicle	-	200	9	4.5	8	4
18 h	28 h	12.5	-	200	6	3	3	1.5
18 h	28 h	EMS 500	-	100	22	22	19	19
4 h	28 h	Vehicle	+	200	7	3.5	3	1.5
4 h	28 h	3.13	+	200	8	4	4	2
4 h	28 h	6.25	+	200	11	5.5	9	4.5
4 h	28 h	12.5	+	200	7	3.5	4	2
4 h	28 h	CPP 0.5	+	1000	17	17	15	15

EMS = ethyl methanesulfonate

CPP = cyclophosphamide

 

Discussion and conclusion:

 

According to the results of the present in vitro cytogenetic study, the test substance did not lead to an increase in the number of structural chromosomal aberrations incl. and excl. gaps either without S-9 mix or after the addition of a metabolizing system in two experiments performed independently of each other selecting different exposure times (4 hours treatment and continuous treatment) and sampling times (18 and 28 hours); the types and frequencies of aberrations were close to the range of that of the concurrent negative control values at both sampling times and in the range of the historical control data.

No increase in the number of cells containing numerical chromosomal aberrations was demonstrated either.

The increase in the frequencies of chromosomal aberrations induced by the positive control agents EMS and CPP clearly demonstrated the sensitivity of the test method and of the metabolic activity of the S-9 mix employed.

 

Thus, under the experimental conditions chosen here, the conclusion is drawn that the test article is neither a clastogenic (chromosome-damaging) nor an aneugenic agent under in vitro conditions using V79 cells.

Conclusions:

Under the experimental conditions chosen here, the conclusion is drawn that the test article is neither a clastogenic (chromosome-damaging) nor an aneugenic agent under in vitro conditions using V79 cells.

Executive summary:

In an GLP and OECD guideline compliant study (No. 473) the test substance was assessed for its potential to induce structural chromosomal aberrations (clastogenic activity) and/or changes in the number of chromosomes (aneugenic activity) in V79 cells in vitro both in the presence and in the absence of a metabolizing system. According to an initial range-finding cytotoxicity test for the determination of the highest experimental doses, the test substance did not exhibit any pronounced toxicity up to the highest possible dose, i.e. 3 000 μg/mL, at which distinct test substance precipitation was observed. However, due to strong test substance precipitation, which interfers with metaphase evaluation, lower doses must be selected and the test groups in bold type were evaluated:

• 1st experiment

4-hour exposure, 18-hour sampling time, without S-9 mix

0; 1.56;3.13; 6.25; 12.5; 25.0; 50.0; 75.0; 100.0 μg/mL

4-hour exposure, 18-hour sampling time, with S-9 mix

0; 1.56;3.13; 6.25; 12.5; 25.0; 50.0; 75.0; 100.0 μg/mL

• 2nd experiment

18-hour exposure, 18-hour sampling time, without S-9 mix

0; 0.78; 1.56;3.13; 6.25; 12.5; 25.0 μg/mL

18-hour exposure, 28-hour sampling time, without S-9 mix

0; 3.13; 6.25;12.5μg/mL

4-hour exposure, 28-hour sampling time, with S-9 mix

0; 1.56;3.13; 6.25; 12.5; 25.0 μg/mL

Doses > 12.5 μg/mL both with and without S-9 mix led to strong precipitation which interferes with evaluation of metaphases. About 2 - 3 hours prior to harvesting the cells, Colcemid was added to arrest cells at a metaphase-like stage of mitosis (c-metaphases). After preparation of the chromosomes and staining with Giemsa, 100 metaphases for each culture in the case of the test substance and vehicle controls, or 50 cells for each culture in the case of the concurrent positive controls, were analyzed for chromosomal aberrations. The negative controls (vehicle controls) gave frequencies of aberrations within the range expected for the V79 cell line. Both of the positive control chemicals, i.e. EMS and cyclophosphamide, led to the expected increase in the number of cells containing structural chromosomal aberrations. On the basis of the results of the present study, the test substance did not cause any increase in the number of structurally aberrant metaphases incl. and excl. gaps at both sampling times either without S-9 mix or after adding a metabolizing system in two experiments performed independently of each other. No increase in the frequency of cells containing numerical aberrations was demonstrated either. Thus, under the experimental conditions of this assay, the test material is considered not to be either a clastogenic or an aneugenic agent under in vitro conditions in V79 cells.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 October - 27 October 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(1997)

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF Aktiengesellschaft, Exerlmental Toxicology and Ecology, 67056 Ludwigshafen

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Physical state: Solid, black powder
- Analytical purity: > 99%
- Storage condition of test material: Room temperature
- Expiration date of the lot/batch: 18 November 2020
- CAS No. Cis: 55034-81-6 Trans: 55034-79-2

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:

S9-mix from male Sprague-Dawley rat livers (treated ip with 500 mg Aroclor 1254/kg bw)

Test concentrations with justification for top dose:

1st experiment: 0; 20; 100; 500; 2 500 and 5 000 µg/plate (SPT);
2nd experiment: 0; 4; 20; 100; 500 and 2 500 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 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

Remarks:

sterility control

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: With S-9 mix: 2-aminoanthracene for all strains; Without S-9 mix: N-methyl-N'-nitro-N-nitrosoguanidine for TA 1535, TA 100; 4-nitro-o-phenylendiamine for TA 98; 9-aminoacridine for TA 1537; 4-nitroquinoline-N-oxide for E. coli WP2 uvrA.

Details on test system and experimental conditions:

METHOD OF APPLICATION: Standard plate test
DURATION
- Exposure duration: 48 - 72 h at 37°C in the dark
NUMBER OF REPLICATIONS: 3

METHOD OF APPLICATION: Preincubation test
DURATION
- Preincubation period: 20 min at 37°C
- Exposure duration: 48 - 72 h
NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
Toxicity detected by a:
- 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 nonmutagenic 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:

no cytotoxicity

Remarks:

no bacteriotoxic effect observed

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test substance was found from about 100 µg/plate onward.
- Toxicity: No bacteriotoxic effect (reduced his or trp background growth, decrease in the number of his+ or trp+ revertants, reduction in the titer) was observed either in the standard plate test or in the preincubation assay.

Results of the Standard Plate Test:

	TA 1535		TA 100		TA 1537		TA 98		WP2uvrA
Dose (µg/plate)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
0	20	17	113	107	10	9	31	32	33	37
20	18	18	106	110	10	10	27	25	33	34
100	18	17	104	104	8	9	28	31	32	35
500	16	16	110	105	8	8	22	36	35	33
2500	15	17	99	107	8	7	20	29	32	33
5000	15	15	111	109	5	7	20	26	28	32
2-AA		139		926		147		653		231
MNNG	690		1091
AAC					487
NOPD							515
4-NQO									603

Results of the Preincubation Test:

	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	109	111	10	10	28	32	34	33
4	18	17	108	109	9	9	27	27	31	30
20	17	17	104	109	9	8	28	27	30	35
100	16	16	104	103	9	9	24	28	30	32
500	15	15	107	109	7	8	28	24	28	27
2500	11	12	103	95	7	6	19	23	25	25
2-AA		130		1059		127		666		231
MNNG	561		904
AAC					467
NOPD							467
4-NQO									537

Controls:

2-AA: 2-aminoanthracene (2.5 µg/plate for all TA strains, 60 µg/plate for WP2uvrA)

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

AAC: 9-aminoacridine (100 µg/plate)

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

4-NQO: 4-nitroquinoline-N-oxide (5 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:

In a GLP compliant bacterial reverse mutation assay according to OECD testing guideline 471, the test substance's mutagenic potential was assessed based on its ability to induce point mutations in selected loci of the tester strains TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA. The substance was tested in a standard plate test and in a preincubation test at dose levels ranging from 20 μg - 5 000 μg/plate and from 4 µg - 2500 µg/plate, respectively. Both tests were performed with and without metabolic activation (Arcolor induced rat liver S9 mix). Precipitation of the test substance was found from about 100 µg/plate onward. No bacteriotoxic effect was observed. An increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either with or without S9 mix. Therefore, the test substance is considered to be not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Bacterial Mutagenicity

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains (Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 98 and Escherichia coli WP2 uvrA) in a reverse mutation assays according to OECD TG 471 and GLP regulations (BASF, 2004; BASF, 2006). In the first Ames assay, the substance was tested at concentrations of 20 µg - 5000 µg/plate with and without metabolic activation (Aroclor-induced rat liver S-9 mix) in the standard plate test and preincubation test. No bacteriotoxic effect or mutagenicity were observed in either test, but test substance precipitation was observed at all tested concentrations.

Therefore, the Ames assay was repeated using a lower range of concentrations: 20 µg - 5000 µg/plate in the standard plate test and 4 µg - 2500 µg/plate in the preincubation test, in the presence and absence of metabolic activation. Under the test conditions of this assay, precipitation of the substance occurred from about 100 µg/plate onward. No bacteriotoxic effect or mutagenicity were observed. Thus, according to the results of both studies, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay under the experimental conditions chosen.

 

Mammalian Mutagenicity

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

An in vitro chromosome aberration assay was conducted in V79 cells according to OECD TG 473 and GLP regulations (BASF, 2006). The substance was assessed for its potential to induce structural chromosomal aberrations (clastogenic activity) and/or changes in the number of chromosomes (aneugenic activity) in V79 cells both in the presence and in the absence of a metabolizing system (Aroclor-induced rat liver S-9 mix). The study comprised two independent experiments. In the first experiment, an exposure time of 4 hours and a sampling time of 18 hours were employed. Doses were between 1.56 µg/mL and 100 µg/mL, with and without S-9 mix. However, due to strong test substance precipitation, which interfers with metaphase evaluation, lower doses were selected for evaluation (3.13; 6.25; 12.5 µg/mL).

The second experiment comprised three assays:

1. 18-hour exposure, 18-hour sampling time, without S-9 mix; doses were 0.78 – 25 µg/mL.


2. 18-hour exposure, 28-hour sampling time, without S-9 mix; doses were 3.13 – 12.5 µg/mL.


3. 4-hour exposure, 28-hour sampling time, with S-9 mix; doses were 1.56 – 25 µg/mL.

Again doses ≥ 12.5 μg/mL led to strong test substance precipitation which could not be evaluated for chromosome aberrations. Both of the positive control chemicals, i.e. ethyl methanesulfonate and cyclophosphamide, led to the expected increase in the number of cells containing structural chromosomal aberrations. On the basis of the results of the present study, the test substance did not cause any increase in the number of structurally aberrant metaphases incl. and excl. gaps at both sampling times either without S-9 mix or after adding a metabolizing system in two experiments performed independently of each other. No increase in the frequency of cells containing numerical aberrations was demonstrated either. Thus, under the experimental conditions of this assay, the substance is considered not to be either a clastogenic or an aneugenic agent under in vitro conditions in V79 cells.

 

Further toxicological data of category members:

The test article belongs to the "perylene based organic pigments" category (see attached document for details on category members and for read across justification). Regarding the genetic toxicity, additional reliable data are available for other category members. All of the studies are taken into account for the evaluation and assessment of the toxicity of the test article.

At least one Ames tests per substance is available for all other category members. 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.

The additional HPRT and CA assays, performed for other category members, each with and without metabolic activation, were also negative and lead no evidence for a mutagenic potential of the test substances.

Justification for classification or non-classification

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

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. No indication of genotoxicity was observed in the Ames test (OECD 471, GLP), the HPRT Test (OECD 476, GLP) and the in vitro chromosome aberration assay (OECD 473, GLP). As a result, the substance is not considered to be classified for mutagenicity under Regulation (EC) No. 1272/2008, as amended for the fourteenth time in Regulation (EC) No. 2020/217.