7,17,28,38-tetraazatridecacyclo[24.16.2.2²,⁵.1⁸,¹².1²⁹,³³.0³,²².0⁴,¹⁹.0⁶,¹⁷.0²³,⁴³.0²⁷,³⁸.0⁴⁰,⁴⁴.0¹⁶,⁴⁶.0³⁷,⁴⁵]octatetraconta-1(42),2(48),3,5(47),6,8,10,12(46),13,15,19,21,23,25,27,29(45),30,32,34,36,40,43-docosaene-18,39-dione; 7,17,28,38-tetraazatridecacyclo[24.16.2.2²,⁵.1⁸,¹².1²⁹,³³.0³,²².0⁴,¹⁹.0⁶,¹⁷.0²³,⁴³.0²⁸,³⁹.0⁴⁰,⁴⁴.0¹⁶,⁴⁶.0³⁷,⁴⁵]octatetraconta-1(42),2(48),3,5(47),6,8,10,12(46),13,15,19,21,23,25,29,31,33,35,37(45),38,40,43-docosaene-18,27-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, 2005, K2

 

HPRT: Read across, 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:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Reason / purpose for cross-reference:

read-across source

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

Reference 2

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
- Expiration date of the lot/batch: 18 November 2020
- CAS No. Cis: 55034-81-6 Trans: 55034-79-2

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 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 29, 2005 - July 15 2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

precipitation occured already at the lowest test concentration.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(July 21, 1997)

Deviations:

yes

Remarks:

(2-aminoanthracene was used as the sole indicator of the efficacy of the S9-mix.)

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)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Physical state: Powder / black
- Analytical purity: 99.9%
- Storage condition of test material: Room temperature
- Expiration date of the lot/batch: unlimited at room temperature

Target gene:

his- and trp-gene

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 µg - 5000 µg/plate (SPT)
20 µg - 5000 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 was the most suitable in comparison to other commonly used vehicles and for which historical control data are available.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see: Details on test system and conditions

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation;

STANDARD PLATE TEST (SPT):
- Salmonella typhimurium:
The experimental procedure of the standard plate test (plate incorporation method) is based an the method of Ames et al. (1975, 1983).
Test tubes containing 2-ml portions of soft agar (overlay agar), which consists of 100 ml agar (0.8% agar + 0.6% NaCI) and 10 ml amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) are kept in a water bath at about 42 - 45°C, and the remaining components are added in the following order:
0.1 ml test solution or vehicle
0.1 ml fresh bacterial culture
0.5 ml S-9 mix (in tests with metabolic activation)
or
0.5 ml phosphate buffer (in tests without metabolic activation)
After mixing, the samples are poured onto Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds.
After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies (his+ revertants) are counted.

- Escherichia coli
The experimental procedure is based on the method of Ames et al. (1975, 1983).
Test tubes containing 2-ml portions of soft agar (overlay agar), which consists of 100 ml agar (0.8% agar + 0.6% NaCI) and 10 ml amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) are kept in a water bath at about 42 - 45°C, and the remaining components are added in the following order:
0.1 ml test solution or vehicle
0.1 ml fresh bacterial culture
0.5 ml S-9 mix (in tests with metabolic activation)
or
0.5 ml phosphate buffer (in tests without metabolic activation)
After mixing, the samples are poured onto minimal agar plates within approx. 30 seconds.
After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies (trp+ revertants) are counted.

PREINCUBATION TEST (PIT):
The experimental procedure is based on the method described by Yahagi et al. (1977) and Matsushima et al. (1980).
0.1 ml test solution or vehicle, 0.1 ml bacterial suspension and 0.5 ml S-9 mix or phosphate buffer are incubated at 37°C for the duration of about 20 minutes using a shaker.
Subsequently, 2 ml of soft agar is added and, after mixing, the samples are poured onto the agar plates within approx. 30 seconds.
After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies are counted.

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

DETERMINATION OF CYTOTOXICITY:
detected by a
- decrease in the number of revertants
- clearing or diminution of the background lawn (= reduced hi-s or trp- background growth)
- reduction in the titer

TITER:
The titer is generally determined only in the experimental parts with S-9 mix both for the negative controls (vehicle only) and for the two highest doses in all experiments.

POSITIVE CONTROLS:
With S-9 mix:
2-aminoanthracene (2-AA): 2.5 µg/plate; dissolved in DMSO; strains: TA 1535, TA 100, TA 1537, TA 98;
60 µg/plate, dissolved in DMSO; strain: Escherichia coli WP2 uvrA

Without S-9 mix:
N-methyl-N'-nitro-N-nitrosoguanidine (MNNG): 5 µg/plate, dissolved in DMSO; TA 1535, TA 100;
4-nitro-o-phenylendiamine (NOPD): 10 µg/plate, dissolved in DMSO; TA 98;
9-aminoacridine (AAC): 100 µg/plate, dissolved in DMSO; TA 1537;
4-nitroquinoline-N-oxide (4-NQO): 5 µg/plate, dissolved in DMSO; E. coli WP2 uvrA

Evaluation criteria:

Acceptance criteria:

Generally, the experiment is considered valid if the following criteria are met:
- The number of revertant colonies in the negative controls was within the normal range of the historical control data for each tester strain.
- The sterility controls revealed no indication of bacterial contamination.
- The positive control articles both with and without S-9 mix induced a significant increase in the number of revertant colonies within the range of the historical control data or above
- The titer of viable bacteria was >/= 10^8/mI.

Assessment 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 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A weak bacteritoxic effect was observed under all test conditions (see additional information on results for details).

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A weak bacteritoxic effect was observed under all test conditions (see additional information on results for details).

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test substance was found from about 20 µg/plate onward. As long as precipitation does not interfere with the colony scoring, 5 mg/plate is generally selected and analyzed (in cases of nontoxic compounds) as the maximum dose even in the case of relatively insoluble test compounds to detect possible mutagenic impurities.

COMPARISON WITH HISTORICAL CONTROL DATA: yes

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A slight decrease in the number revertants was occasionally observed in the standard plate test at 5000 µg/plate.
In the preincubation assay bacteriotoxicity (slight decrease in the number of revertants and / or reduction in the titer) was observed depending on the strain and test conditions from about 2 500 µg/plate onward.

1st experiment: Standard plate test (20 - 5000 µg/plate)
Strain	Metabolic activation system	mean his^+/trp^+revertant colonies (negative control)	maximum revertant factor (conc. (µg/plate))	dose dependency	Assessment	maximum revertant factor (positive control)
TA 98	no	28	1.0 (20)	no	negative	16.4 (NOPD)
	yes	35	1.0 (20/500/2500)	no	negative	15.6 (2-AA)
TA 100	no	112	1.0 (20/100/500)	no	negative	6.7 (MNNG)
	yes	108	1.0 (20/100/500/2500/5000)	no	negative	8.6 (2-AA)
TA 1537	no	11	0.9 (20)	no	negative	38.3 (AAC)
	yes	9	1.3 (20)	no	negative	13.9 (2-AA)
TA 1535	no	16	1.0 (100)	no	negative	36.6 (MNNG)
	yes	17	1.1 (100/500)	no	negative	7.7 (2-AA)
E. coli WP2 uvrA	no	35	0.9 (100/500/2500/5000)	no	negative	17.6 (4-NQO)
	yes	36	1.4 (5000)	no	negative	6.0 (2-AA)
2nd experiment: Preincubation test (20 - 5000 µg/plate)
Strain	Metabolic activation system	mean his^+/trp^+revertant colonies (negative control)	maximum revertant factor (conc. (µg/plate))	dose dependency	Assessment	maximum revertant factor (positive control)
TA 98	no	20	1.3 (20)	no	negative	28 (NOPD)
	yes	35	1.0 (100)	no	negative	17.1 (2-AA)
TA 100	no	114	1.0 (20/100/2500/5000)	no	negative	7.5 (MNNG)
	yes	115	1.0 (20/100/500)	no	negative	6.7 (2-AA)
TA 1537	no	13	0.6 (100/500/5000)	no	negative	46.6 (AAC)
	yes	9	1.1 (500)	no	negative	13.4 (2-AA)
TA 1535	no	14	1.0 (100)	no	negative	61.9 (MNNG)
	yes	12	1.1 (2500)	no	negative	27.7 (2-AA)
E. coli WP2 uvrA	no	43	1.0 (20)	no	negative	19.3 (4-NQO)
	yes	43	0.6 (20/100/500)	no	negative	5.4 (2-AA)
Precipitation of the test substance was found from about 20 µg/plate onward.
2-AA: 2-aminoanthracene
MNNG: N-methyl-N'-nitro-N-nitrosoguanidine
NOPD: 4-nitro-o-phenylendiamine
AAC: 9-aminoacridine
4-NQO: 4-nitroquinoline-N-oxide

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 reverse mutation assay according to OECD guideline 471 the test article was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains. Tester strains were Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA.

The test was conducted at a dose range from 20 µg - 5 000 µg/plate in the standard plate test as well as in the preincubation test, both with and without metabolic activation (Aroclor-induced rat liver S-9 mix).

Precipitation of the test substance occurred from about 20 µg/plate onward. A weak bacteritoxic effect was observed under all test conditions. 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 S9 mix or after the addition of a metabolizing system. Therefore, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay under the experimental conditions chosen here.

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 Nov 2005 - 14 Feb 2005

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)

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)

Remarks:

BASF AG, Experimental Toxicology and Ecology, Ludwigshafen, Germany

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

- Physical state: Powder / black
- Storage condition of test material: Room temperature
- Lot/batch No.: Lot 2005-01
- Expiration date of the lot/batch: unlimited at 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 (10000 IU /10000 µg/mL)
- 1% (v/v) amphotericin B (250 µg/mL)
During exposure to the test substance (4-hour treatment), MEM medium was used without FCS supplementation.

- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Checked for plating efficiency (= colony forming ability) incl. vital staining

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9-mix

Test concentrations with justification for top dose:

1st experiment: 1.563, 3.125, 6.25, 12.5, 25, 50, 75 µg/mL (with and without metabolic activation)
2st experiment: 0.781, 1.563, 3.125, 6.25, 12.5 µg/mL (without metabolic activation); 1.563, 3.125, 6.25, 12.5, 25 µg/mL (with metabolic activation);

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 the V79 in vitro cytogenetic test and for which historical control data are available.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: (500 µg/mL ethylmethanesulfonate (EMS) without S9; 0.5 µg/mL cyclophosphamide (CPP) with S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

30000 - 80000 cells were seeded in each chamber of Quadriperm dishes. Two chambers of a Quadriperm dish were used for one test culture.

DURATION

- Preincubation period: 24 - 30 hours

1st experiment:
exposure: 4 h; sampling 18 h (1.563, 3.125, 6.25, 12.5, 25, 50, 75 µg/mL + controls with and without metabolic activation);

2nd experiment:
exposure: 18 h; sampling: 18 h (0.781, 1.563, 3.125, 6.25, 12.5 µg/mL + controls without metabolic activation);
exposure: 18 h; sampling: 28 h (3.125, 6.25 µg/mL + control without metabolic activation);
exposure: 4 h; sampling: 28 h ( 1.563, 3.125, 6.25, 12.5, 25 µg/mL + control with metabolic activation);

SPINDLE INHIBITOR (cytogenetic assays): 0.2 µg Colcemid/mL culture medium
STAIN (for cytogenetic assays): solution of Giemsa and Titrisol (15 mL Giemsa, 185 mL Titrisol pH 7.2)

NUMBER OF REPLICATIONS: duplicate

NUMBER OF CELLS EVALUATED: 200 metaphases were analyzed normally (100 of each culture); However, due to a clear increase in chromosomally damaged cells, the number of metaphases analyzed was reduced from the planned 200 mitoses to 100 cells:
1st experiment: 0.5 µg/mL CPP;
2nd experiment: 500 µg/mL EMS, 0.5 µg/mL CPP;

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cell counts

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- Other: About 3 hours and 16 - 18 hours after test substance treatment, cultures of all test groups were checked for ceII morphology, which is an indication of attachment of the cells to the slides

Evaluation criteria:

Acceptance criteria:

The V79 in vitro cytogenetic assay is considered valid if the following criteria are met:
- The quality of the slides must allow the identification and evaluation of a sufficient number of analyzable metaphases.
- The numbers of cells with structural / numerical aberrations in the negative control (vehicle control) has to be within the normal range of the historical negative control data
- The positive control chemicals both with and without S-9 mix have to induce a significant increase in the number of cells with structural aberrations

Assessment criteria:

The test chemical will be assessed as "positive" in this assay if the following criteria are met:
- A dose-related and reproducible significant increase in the number of cells with structural / numerical chromosomal aberrations.
- The number of aberrant cells exceeds both the concurrent negative control range and the highest value of the negative historical control range.
A test substance generally will be considered as "negative" if the following criteria are met:
- The number of cells with structural / numerical aberrations in the dose groups is not significantly above the concurrent negative control and is within the historical control data.

Statistics:

The statistical evaluation of the data was carried out using the MUCHAN program system. 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 are 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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH / osmolality: Osmolarity and pH values were not influenced by test substance treatment.
- Precipitation:
1st experiment: evaluation was not possible due to strong test substance precipitation which interferes with metaphase evaluation at a concentration of 12.5 µg/mL and above (with and without metabolic activation)
2nd experiment: evaluation was not possible due to strong test substance precipitation which interferes with metaphase evaluation at a concentration of 12.5 µg/mL (without metabolic activation) and of 12.5 µg/mL and above (with metabolic activation), respectively

RANGE-FINDING/SCREENING STUDIES: The test doses/concentrations were selected on the basis of the non-GLP range finding part of the study. The doses for the 1st experiment (18-hour sampling time) were determined from appropriate range-finding cytotoxicity tests with cultures exposed for the duration of 4 hours to a wide dose range of the test article, i.e. 1 µg/mL - 2 500 µg/mL culture medium both without S-9 mix and after adding a metabolizing system. In these pretests, various parameters were checked for all or at least some selected doses. Up to a dose of 2500 µg/mL, at which distinct test substance precipitation was observed, the test substance did not exhibit any toxic effects after a treatment time of 4 hours. Doses > 2500 µg/mL led to an inhomogenous mass which could not be administered any longer. On the basis of the findings from the pretests, due to strong test substance precipitation which interferes with metaphase evaluation, a dose range of only 1.563 µg/mL - 75 µg/mL was selected without S-9 mix and with metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA: yes

ADDITIONAL INFORMATION ON CYTOTOXICITY: 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. According to the results of the cell count, no growth inhibition was observed under any of the experimental conditions. CeII attachment was not influenced at any dose evaluated for structural chromosomal aberrations.

1st experiment
without S9 mix: 4 h treatment, 18 h sampling
Dose	No. Of metaphases	Metaphases with aberrations
		Incl. Gaps		Excl. Gaps		Exchanges		Mul. Aber.		Chr. Dis.		Aneupl.		Polypl.		Endopl.
		n	%	n	%	n	%	n	%	n	%	n	%	n	%	n	%
DMSO	200	7	3.5	5	2.5	1	0.5	0	0	0	0	1	0.5	4	2	0	0
1.563 µg/mL	200	5	2.5	3	1.5	2	1	0	0	0	0	0	0	1	0.5	0	0
3.125 µg/mL	200	10	5	9	4.5	6	3	0	0	0	0	0	0	0	0	0	0
6.25 µg/mL	200	6	3	1	0.5	0	0	0	0	0	0	0	0	0	0	0	0
EMS 500 µg/mL	200	20	10*	17	8.5*	11	5.5**	0	0	0	0	0	0	0	0	0	0
*p</= 0.05
**p</= 0.01
Evaluation not possible due to strong test substance precipitation which interferes with metaphase evaluation at concentrations of 12.5, 25, 50 and 75 µg/mL
with S9 mix: 4 h treatment, 18 h sampling
Dose	No. Of metaphases	Metaphases with aberrations
		Incl. Gaps		Excl. Gaps		Exchanges		Mul. Aber.		Chr. Dis.		Aneupl.		Polypl.		Endopl.
		n	%	n	%	n	%	n	%	n	%	n	%	n	%	n	%
DMSO	200	11	5.5	4	2	1	0.5	0	0	0	0	0	0	1	0.5	3	1.5
1.563 µg/mL	200	9	4.5	7	3.5	3	1.5	0	0	0	0	1	0.5	2	1	0	0
3.125 µg/mL	200	9	4.5	1	0.5	0	0	0	0	0	0	0	0	1	0.5	1	0.5
6.25 µg/mL	200	5	2.5	3	1.5	1	0.5	0	0	0	0	0	0	3	1.5	1	0.5
CPP 0.5 µg/mL	100	23	23**	20	20**	14	14**	1	1	0	0	0	0	0	0	0	0
**p</= 0.01
Evaluation not possible due to strong test substance precipitation which interferes with metaphase evaluation at concentrations of 12.5, 25, 50 and 75 µg/mL
2nd experiment
without S9 mix: 18 h treatment, 18 h sampling
Dose	No. Of metaphases	Metaphases with aberrations
		Incl. Gaps		Excl. Gaps		Exchanges		Mul. Aber.		Chr. Dis.		Aneupl.		Polypl.		Endopl.
		n	%	n	%	n	%	n	%	n	%	n	%	n	%	n	%
DMSO	200	7	3.5	1	0.5	0	0	0	0	0	0	0	0	5	2.4	0	0
1.563 µg/mL	200	11	5.5	3	1.5	0	0	0	0	0	0	0	0	3	1.5	0	0
3.125 µg/mL	200	8	4	5	2.5	3	1.5	0	0	0	0	0	0	2	1	0	0
6.25 µg/mL	200	9	4.5	2	1	0	0	0	0	0	0	0	0	1	0.5	0	0
EMS 500 µg/mL	100	23	23**	21	21**	11	11**	0	0	0	0	0	0	0	0	0	0
**p</= 0.01
Evaluation not possible due to strong test substance precipitation which interferes with metaphase evaluation at concentrations of 12.5 µg/mL (additionally 0.781 µg/mL: no evaluation)
without S9 mix: 18 h treatment, 28 h sampling
Dose	No. Of metaphases	Metaphases with aberrations
		Incl. Gaps		Excl. Gaps		Exchanges		Mul. Aber.		Chr. Dis.		Aneupl.		Polypl.		Endopl.
		n	%	n	%	n	%	n	%	n	%	n	%	n	%	n	%
DMSO	200	9	4.5	3	1.5	0	0	0	0	0	0	0	0	1	0.5	0	0
6.25 µg/mL	200	6	3	1	0.5	1	0.5	0	0	0	0	0	0	0	0	0	0
EMS 500 µg/mL	100	17	17**	17	17**	14	14**	0	0	0	0	0	0	0	0	0	0
**p</= 0.01
03.125 µg/mL: no evaluation
with S9 mix: 4 h treatment, 28 h sampling
Dose	No. Of metaphases	Metaphases with aberrations
		Incl. Gaps		Excl. Gaps		Exchanges		Mul. Aber.		Chr. Dis.		Aneupl.		Polypl.		Endopl.
		n	%	n	%	n	%	n	%	n	%	n	%	n	%	n	%
DMSO	200	8	4	4	2	3	1.5	0	0	0	0	0	0	3	1.5	0	0
1.563 µg/mL	200	9	4.5	7	3.5	3	1.5	0	0	0	0	0	0	2	1	0	0
3.125 µg/mL	200	6	3	2	1	2	1	0	0	0	0	0	0	1	0.5	0	0
6.25 µg/mL	200	8	4	3	1.5	2	1	0	0	0	0	0	0	3	1.5	1	0.5
CPP 0.5 µg/mL	100	21	21**	20	20**	15	15**	0	0	0	0	0	0	0	0	0	0
**p</= 0.01
Evaluation not possible due to strong test substance precipitation which interferes with metaphase evaluation at concentrations of 12.5 and 25 µg/mL

Conclusions:

Under the experimental conditions of this assay, the test substance is considered not to be either a clastogenic or an aneugenic agent under in vitro conditions in V79 cells.

Executive summary:

In a GLP-compliant chromosome aberration study following OECD guideline 473, the test substance was assessed for its potential to induce structural chromosomal aberrations 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. 2 500 µg/mL, at which distinct test substance precipitation was observed. Doses > 2 500 µg/mL led to an inhomogeneous mass which could not be administered any longer. Due to strong test substance precipitation, which interferes 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 5-9 mix: 0; 1.563; 3.125; 6.25; 12.5; 25.0; 50.0; 75.0 µg/mL

4-hour exposure, 18-hour sampling time, with S-9 mix: 0; 1.563; 3.125; 6.25; 12.5; 25.0; 50.0; 75.0 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 the evaluation of metaphases.

 

2nd experiment:

For confirmation of the results of the 1st experiment including continuous exposure and a second sampling time, the following doses were tested and the test groups in bold type were evaluated:

18-hour exposure, 18-hour sampling time, without 5-9 mix: 0; 0.781; 1.563; 3.125; 6.25; 12.5 µg/mL

18-hour exposure, 28-hour sampling time, without 5-9 mix; 0; 3.125; 6.25 µg/mL

4-hour exposure, 28-hour sampling time, with 5-9 mix: 0; 1.563; 3.125; 6.25; 12.5; 25.0 µg/mL

Again, doses >12.5 µg/ml led to strong precipitation which interferes with the 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 relevant increase in the number of structurally aberrant metaphases mcl. 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 article is considered not to be either a clastogenic or an aneugenic agent under in vitro conditions in V79 cells.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Bacterial Mutagenicity

In a GLP-compliant reverse mutation assay according to OECD guideline 471 the test article was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains (BASF, 2005). Tester strains were Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA. The test was conducted at a dose range from 20 µg - 5 000 µg/plate in the standard plate test as well as in the preincubation test, both with and without metabolic activation (Aroclor-induced rat liver S-9 mix). Precipitation of the test substance occurred from about 20 µg/plate onward. A weak bacteritoxic effect was observed under all test conditions. 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 S9 mix or after the addition of a metabolizing system. Therefore, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay under the experimental conditions chosen here.

A second reliable Ames test (standard plate and preincubation test) with the same study design as the first one confirmed the negative result (BASF, 2004).

 

Mammalian Mutagenicity

As no data for the test substance is available regarding mutagenicity in mammalian cells a read-across to the category member EC 479-300-2 was performed.

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

A chromosomal aberration test in Chinese hamster lung fibroblasts (V79) with and without metabolic activation (metabolic activation: Aroclor 1254-induced rat liver S-9 mix) was conducted according to OECD 473 and under GLP (reliable without restrictions). In the first experiment (exposure: 4 h; sampling 18 h), the test substance was tested up to 75 µg/ml with and without metabolic activation. In the second experiment, the maximal concentration was 12.5 µg/ml (exposure: 18 h; sampling: 18 h; without metabolic activation), 6.25 µg/ml (exposure: 18 h; sampling: 28 h, without metabolic activation) or 25 µg/ml (exposure: 4 h; sampling: 28 h; with metabolic activation). The test was negative, and no cytotoxicity was detected (BASF, 2006). 

 

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.