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

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

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

Test material

Test material information

Constituent 1

Test material form:

solid: nanoform, no surface treatment

Details on test material:

- State of aggregation: solid, powder
- Particle size distribution (TEM): 30.3 nm (D50)
- Mass median aerodynamic diameter (MMAD): not specified
- Geometric standard deviation (GSD): not specified
- Shape of particles: spherical
- Surface area of particles: 16.8 m²/g
- Crystal structure: crystalline
- Coating: no
- Surface properties: not applicable
- Density: 1515 kg/m³ at 20°C
- Moisture content: refer to IUCLID chapter 1
- Residual solvent: refer to IUCLID chapter 1
- Activation: not applicable
- Stabilisation: not applicable

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

Method

Target gene:

HPRT (hypoxanthine-guanine phosphoribosyl transferase)

Species / strain

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).

Controls

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.

Results and discussion

Test results

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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.