2,9-bis(2-phenylethyl)anthra[2,1,9-def:6,5,10-d'e'f']diisoquinoline-1,3,8,10(2H,9H)-tetrone

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:

04 May 2020 - 13 Jul 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

Version / remarks:

29 Jul 2016

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

Aug 1998

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Test material

Test material information

Constituent 1

Reference substance name:

2,9-bis(3,5-dimethylphenyl)anthra[2,1,9-def:6,5,10-d'e'f']diisoquinoline-1,3,8,10(2H,9H)-tetrone

EC Number:

225-590-9

EC Name:

2,9-bis(3,5-dimethylphenyl)anthra[2,1,9-def:6,5,10-d'e'f']diisoquinoline-1,3,8,10(2H,9H)-tetrone

Cas Number:

4948-15-6

Molecular formula:

C40H26N2O4

IUPAC Name:

2,9-bis(3,5-dimethylphenyl)isoquino[4',5',6':6,5,10]anthra[2,1,9-def]isoquinoline-1,3,8,10(2H,9H)-tetrone

Test material form:

solid: nanoform, no surface treatment

Details on test material:

- State of aggregation: solid, powder
- Particle size distribution (TEM): 18.0 nm (D50)
- Mass median aerodynamic diameter (MMAD): not specified
- Geometric standard deviation (GSD): not specified
- Shape of particles: spherical
- Surface area of particles: 76 m²/g
- Crystal structure: crystalline
- Coating: no
- Surface properties: not applicable
- Density: 1415 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:

Batch identification: 100071P040
CAS No.: 4948-15-6
Content: Sum.: 99.7 g/100 g
Date of production: 21 Dec 2010
Date of expiry: 30 Mar 2023
Molecular weight: 598.66 g/mol
Physical state, appearance: Solid, orange-red
Mass-specific surface area (BET): 97.1 m²/g
Storage conditions: Room temperature
Homogeneity: The homogeneity of the test substance was ensured by mixing before preparation of the test substance preparations.
Storage stability: The stability of the test substance under storage conditions was guaranteed until 30 Mar 2023 as indicated by the sponsor, and the sponsor holds this responsibility.

Method

Target gene:

hprt

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: CHO (Chinese hamster ovary)
- Suitability of cells: not specified

For cell lines:
- Absence of Mycoplasma contamination: each batch checked
- Number of passages if applicable: at least 2 passages before experiment; a further passage to prepare test cultures
- Methods for maintenance in cell culture: Cells were grown with 5% (v/v) CO2 at 37°C and ≥ 90% relative humidity up to approximate confluence and subcultured twice weekly (routine passage in 75 cm² plastic flasks).
- Cell cycle length, doubling time or proliferation index : high proliferation rate (doubling time of about 12 - 16 hours)
- Modal number of chromosomes: karyotype with a modal number of 20 chromosomes
- Periodically checked for karyotype stability: not specified
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- All media were supplemented with penicillin/streptomycin (100 IU / 100 μg/mL), amphotericine B (2.50 μg/mL)
- Culture medium/Treatment medium(without S9 mix): Ham's F12 medium with stable glutamine, hypoxanthine, 10% (v/v) fetal calf serum (FCS)
- Treatment medium (with S9 mix): Ham's F12 medium with stable glutamine, hypoxanthine
- Pretreatment medium ("HAT" medium): Ham's F12 medium with hypoxanthine (136 µg/mL), aminopterin (1.8 µg/mL), thymidine (38.8 µg/mL), 10% (v/v) FCS
- Selection medium ("TG" medium): Ham's F12 medium with stable glutamine, 6-thioguanine (10 μg/mL), 10% (v/v) FCS

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9
- method of preparation of S9 mix
- concentration or volume of S9 mix and S9 in the final culture medium
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability)

The S9 fraction was prepared according to Ames et al. (1975):
- At least 5 male Wistar rats [Crl:WI(Han)] (200 - 300 g; Charles River Laboratories Germany GmbH) received 80 mg/kg b.w. phenobarbital i.p. and β-naphthoflavone orally each on three consecutive days.
- During this time, the animals were housed in polycarbonate cages: central air conditioning with a fixed range of temperature of 20 - 24°C and a fixed relative humidity of 45 - 65%. The day/night rhythm was 12 hours: light from 6 am – 6 pm and darkness from 6 pm – 6 am. Standardized pelleted feed and drinking water from bottles were available ad libitum.
- 24 hours after the last administration, the rats were sacrificed and the livers were prepared using sterile solvents and glassware at a temperature of +4°C.
- The livers were weighed and washed in a weight-equivalent volume of a 150 mM KCl solution and homogenized in three volumes of KCl solution. After centrifugation of the homogenate at 9000 x g for 10 minutes at +4°C, 5 mL portions of the supernatant (S9 fraction) were stored at -70°C to -80°C.
- The S9 mix was prepared freshly prior to each experiment (Ames, 1975). For this purpose, a sufficient amount of S9 fraction was thawed at room temperature; 1 part S9 fraction was mixed with 9 parts S9 supplement (cofactors) in the pre-experiment and main experiments. This preparation, the S9 mix (10% S9 fraction), was kept on ice until used. The concentrations of the cofactors in the S9 mix were: 8 mM MgCl2, 33 mM KCL, 5 mM glucose-6-phosphate, 4 mM NADP, 15 mM phosphate buffer (pH 7.4).
- The phosphate buffer (DeMarini, 1989) is prepared by mixing a Na2HPO4 solution with a NaH2PO4 solution in a ratio of about 4:1.

Ames, B.N. et al. (1975): Methods for detecting carcinogens and mutagens with the Salmonella/mammalian microsome mutagenicity test. Mut. Res. 31, 347-364.
DeMarini, D.M. et al. (1989): Cytotoxicity and effect on mutagenicity of buffers in a microsuspension assay. Ter. Carc. Mut. 9, 287-295.

Test concentrations with justification for top dose:

Based on the data and the observations from the pre-test and taking into account the current
guidelines, the following doses were selected in this study:
Dose selection (with and without S9 mix), 4 h exposure: 0.05, 0.08, 0.15, 0.26, 0.48, 0.86, 1.54, 2.78, 5.00 µg/mL

The highest tested concentration (5.00 μg/mL) was based on test substance precipitation in culture medium.

Vehicle / solvent:

Due to the insolubility of the test substance in culture medium, acetone was selected as the vehicle, which had been demonstrated to be suitable in the CHO/HPRT assay and for which historical data are available. The final concentration of the vehicle acetone in culture medium was 1% (v/v).

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

Time schedule:
- Day 1: Seeding of the cells pretreated with "HAT" medium: in 300 cm² flasks (20x10^6 cells in 40 mL)
- Day 2: Test substance incubation (20 – 24 hours after seeding); exposure period (4 hours); removal of test substance by intense washing; 1st passage of the treated cells in 175 cm² flasks (2x10^6 cells in 20 mL medium) and seeding of the cloning efficiency 1 (survival) in 60 mm petri dishes (200 cells in 5 mL medium).
- Day 5: 2nd passage of the treated cells (seeding of 2x10^6 cells in 20mL medium)
- Day 7 - 9: Drying, fixation, staining and counting of the cloning efficiency 1; 3rd passage of the treated cells; addition of selection medium ("TG" medium); and seeding of the cloning efficiency 2 (viability)
- From day 16: Drying, fixation, staining and counting of the selected colonies and cloning efficiency 2

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 2
- Number of independent experiments: 1

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 20x10^6 cells in 40 mL
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Attachment period of the cells: 20 - 24 hours
- Exposure duration/duration of treatment: 4 hours

FOR GENE MUTATION:
- Expression time: 7-9 days (from day 2)
- Selection time: 6-7 days (from day 7 to 9)
- Selective agent: 6-thioguanine (10 μg/mL)
- Fixation time: from day 7-9 and 16
- Each test group were fixed with methanol, stained with Giemsa and counted.

METHODS FOR MEASUREMENT OF CYTOTOXICITY

- Cloning efficiency 1: For the determination of the influence of the test substance after the exposure period, 200 cells per concentration were reserved from the treated cells and were seeded in petri dishes (60 mm diameter) and coated with 5 mL Ham's F12 medium incl. 10% (v/v) FCS in parallel to the 1st passage directly after test substance incubation.
- Cloning efficiency 2: For the determination of the mutation rate after the expression period, two aliquots of 200 cells each were reserved from the transfer into selection medium (after 7 – 9 days) and seeded in two petri dishes (60 mm diameter) containing 5 mL Ham's F12 medium incl. 10% (v/v) FCS.
- In all cases, after seeding the flasks or petri dishes were incubated for 5 - 8 days to form colonies. These colonies were fixed, stained and counted.

The cloning efficiency (CE, %) was calculated for each test group as follows:

total number of colonies in the test group
CEabsolute = ————————————————————— x 100
total number of seeded cells in the test group

CEabsolute of the test group
CErelative = —————————————— x 100
CEabsolute of the vehicle control


The number of colonies in every petri dish was counted and recorded. Using the formula above the values of absolute cloning efficiencies (CEabsolute, CE1 absolute and/or CE2 absolute) were calculated. Based on these values the relative cloning efficiencies (CErelative, CE1 relative and/or CE2 relative) of the test groups were calculated and reported as a percentage of the respective CEabsolute value of the corresponding vehicle control (vehicle control = 100%).
In addition, with regard to cell loss while exposure period, relative survival (RS) is calculated based on CE of cells plated immediatedly after treatment adjusted by any loss of cells during treatment as compared with adjusted cloning efficiency in vehicle controls.

number of cells at the end of treatment
Adjusted CE = —————————————————— x CE1
number of seeded cells

adjusted CE of the test group
RS = ———————————————————— x 100
adjusted CE of the vehicle control


METHODS FOR MEASUREMENTS OF GENOTOXICIY

The number of colonies in each flask was counted and recorded. The sum of the mutant colony counts within each test group was subsequently normalized per every 10^6 cells seeded. The uncorrected mutant frequency (MFuncorr.) per 10^6 cells was calculated for each test group as follows:

total number of mutant colonies
MFuncorr. = —————————————–— x 10^6
number of seeded cells

The uncorrected mutant frequency was corrected with the absolute cloning efficiency 2 for each test group to get the corrected mutant frequency (MFcorr.):

MFuncorr.
MFcorr. = —–——–— x 100
CE2 absolute

OTHER:
- Check or determination of further parameters: pH, osmolality, solubility, cell morpology

Evaluation criteria:

Acceptance criteria:

- The HPRT assay is considered valid if the following criteria are met:
• The absolute cloning efficiencies of the vehicle controls should not be less than 50% (with and without S9 mix).
• The background mutant frequency in the vehicle controls should be within our historical negative control data range (95% control limit). Weak outliers can be judged acceptable if there is no evidence that the test system is not “under control”.
• Concurrent positive controls both with and without S9 mix should induce responses that are compatible with those generated in the historical positive control data base and produce a statistically significant increase in mutant frequencies compared with the concurrent vehicle control.


Assessment criteria:

- A test substance is considered to be clearly positive if all following criteria are met:
• A statistically significant increase in mutant frequencies is obtained.
• A dose-related increase in mutant frequencies is observed.
• The corrected mutation frequencies (MFcorr.) exceeds both the concurrent vehicle control value and the range of our laboratory’s historical negative control data (95% control limit).

- Isolated increases of mutant frequencies above our historical negative control range or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.

- A test substance is considered to be clearly negative if the following criteria are met:
• Neither a statistically significant nor dose-related increase in the corrected mutation
frequencies is observed under any experimental condition.
• The corrected mutation frequencies in all treated test groups is close to the concurrent
vehicle control value and within the range of our laboratory’s historical negative control data
(95% control limit).

Statistics:

A linear dose-response was evaluated by testing for linear trend. The dependent variable was the corrected mutant frequency and the independent variable was the dose.
The calculation was performed using EXCEL function RGP.
The used model is one of the proposed models of the International Workshop on Genotoxicity
Test procedures Workgroup Report (Moore, 2003).
A pair-wise comparison of each test group with the control group was carried out using Fisher's exact test with Bonferroni-Holm correction (Holm, 1979 and Siegel, 1956). The calculation was performed using EXCEL function HYPGEOM.VERT.
If the results of these tests were statistically significant compared with the respective vehicle control, labels (s p ≤ 0.05) are printed in the tables.
However, both, biological and statistical significance are considered together.


- Moore, M.M. et al. (2003) Mouse Lymphoma Thymidine Kinase Gene Mutation Assay: International Workshop on Genotoxicity Tests (IWGT) Workgroup Report – Plymouth, UK 2002. Mut. Res. 540, 127–140.
- Holm, S. (1979) A Simple Sequentially Rejective Multiple Test Procedure. Scand J Statist 6, 65-70.
- Siegel, S. (1956) Nonparametric statistics for the behavioral sciences. New York, NY, US: McGraw-Hill

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

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

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: not affected
- Data on osmolality: not affected
- Possibility of evaporation from medium: no
- Water solubility: insoluble in water and cell culture medium
- Precipitation and time of the determination: test substance precipitation was observed at 1.25 μg/mL and above after 4 hours in the absence and presence of S9 mix
- Cell morphology and attachment of the cells: not adversely influenced (grade > 2) in any test group

RANGE-FINDING/SCREENING STUDIES:
An initial range-finding cytotoxicity test for the determination of the experimental doses was conducted.
The pre-test was performed following the method described for the main experiment. The relative survival (RS) was determined as a toxicity indicator for dose selection and various parameters were checked for all, or at least some, selected doses.
In the pre-test for toxicity based on the solubility properties of the test substance 10.00 μg/mL (approx. 0.02 mM) was used as top concentration both with and without S9 mix at 4 hour exposure time.

STUDY RESULTS
- The mutation frequencies of the vehicle control groups were within our historical negative control data range (95% control limit) and, thus, fulfilled the acceptance criteria of this study.
- The increase in the frequencies of mutant colonies induced by the positive control substances EMS and DMBA clearly demonstrated the sensitivity of the test method and/or of the metabolic activity of the S9 mix employed.

For all test methods and criteria for data analysis and interpretation:
- At least four concentrations were evaluated to describe a possible dose response relationship.
- Statistical analysis; statistical significance for p ≤ 0.05
- see table 1 in section "Any other information on results incl. tables"

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements and genotoxicity results: see table 2 in section "Any other information on results incl. tables"

HISTORICAL CONTROL DATA
please see table 3 and 4 in section "Any other information on results incl. tables"

Any other information on results incl. tables

Table 1: Linear trend-test

 

Linear trend-test	Slope*	One-sided p-value*
Corrected Mutation frequency without S9 mix	-0.71162	0.7360
Corrected Mutation frequency with S9 mix	-1.32571	0.8616

* The linear trend-test testing for an increased mutant frequency is significant (significance level of 5%), if the one-sided p-value is lower than 0.05 and the slope is greater than 0.

 

Table 2: Summary of results

 

Exp.	Exposure period	Test groups	S9 mix	Prec.*	Genotoxicity** MFcorr.	Cytotoxicity***
	[h]	[µg/mL]			[per 10^6 cells]	RS [%]	CE2 [%]
1	4	VC^1	-	n.d.	1.05	100.0	100.0
		0.05	-	-	n.c.	125.6	n.c.
		0.08	-	-	n.c.	134.3	n.c.
		0.15	-	-	n.c.	135.3	n.c.
		0.26	-	-	3.39	128.5	103.5
		0.48	-	-	0.00	124.5	135.8
		0.86	-	-	0.68	132.5	103.5
		1.54	-	+	0.61	128.7	114.7
		2.78	-	+	n.c.^1	n.c.^1	n.c.^1
		5.00	-	+	n.c.^1	n.c.^1	n.c.^1
		PC^2	-	n.d	42.12^s	113.2	102.5
1	4	VC^1	+	n.d.	1.42	100.0	100.0
		0.05	+	-	n.c.	91.9	n.c.
		0.08	+	-	n.c.	89.1	n.c.
		0.15	+	-	n.c.	103.8	n.c.
		0.26	+	-	3.13	107.9	102.5
		0.48	+	-	1.69	68.1	105.3
		0.86	+	-	1.53	96.1	92.9
		1.54	+	+	0.72	82.6	98.9
		2.78	+	+	n.c.^1	n.c.^1	n.c.^1
		5.00	+	+	n.c.^1	n.c.^1	n.c.^1
		PC^3	+	n.d.	48.52^s	72.6	84.3

 

* Microscopically visible precipitation in culture medium at the end of exposure period

** Mutant frequency MFcorr.: mutant colonies per 10⁶ cells corrected with the CE2 value

*** Cloning efficiency related to the respective vehicle control

^s Mutant frequency statistically significantly higher than corresponding control values (p ≤ 0.05)

n.c. Culture was not continued since a minimum of only four analysable concentrations is required

n.c.¹ Culture was not continued since only one concentration beyond the solubility limit is required

n.d. Not determined

VC vehicle control

PC positive control

¹ Acetone 1% (v/v) ² EMS 400 μg/mL ³ DMBA 1.25 μg/mL

 

 

Table 3: HISTORICAL NEGATIVE CONTROL DATA

Summary (all vehicles)

Period: March 2016 - December 2019

	Without S9 mix all vehicles*	With S9 mix all vehicles*
Corrected Mutant Frequency**
Exposure period	4 hrs	4 hrs
Mean	2.76	2.93
Minimum	0.00	0.00
Maximum	8.00	9.93
Standard Deviation	1.73	2.09
95% Lower Control Limit	0.00	0.00
95% Upper Control Limit	6.21	7.08
No. of Experiments	130	132

* = culture medium, water 10% (v/v), DMSO 1% (v/v), acetone 1% (v/v)

** = mutant frequency (per 1 million cells) corrected with the cloning efficiency at the end of the expression period (CE2)

 

 

Table 4: HISTORICAL POSITIVE CONTROL DATA

Summary (all vehicles)

Period: March 2016 - December 2019

	Without S9 mix 400 µg/mL ethyl methanesulfonate (EMS)	With S9 mix 1.25 µg/mL 7,12-Dimethylbenz[a]anthracene (DMBA)
	Corrected Mutant Frequency*	Corrected Mutant Frequency*
Exposure period	4 hrs	4 hrs
Mean	160.94	126.62
Minimum	42.47	21.52
Maximum	438.29	270.48
Standard Deviation	73.52	54.78
No. of Experiments	124	129

* = mutant frequency (per 1 million cells) corrected with the cloning efficiency at the end of the expression period (CE2)

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions of this study, the test substance is not mutagenic in an in vitro mammalian cell gene mutation test (HPRT-locus) in absence and in the presence of metabolic activation.

Executive summary:

The test substance was evaluated for genotoxic potential in a HPRT locus assay using CHO cells according to OECD TG 476 (GLP compliant). In one experiment a dose range from 0.05 to 5 µg/ml was tested, both with and without the addition of liver S9 mix from phenobarbital and β-naphthoflavone induced rats. Based on the results of the present study, the test substance did not cause any biologically relevant increase in the mutant frequencies either without or after the addition of the metabolizing system (S9 mix). No cytotoxicity could be observed, but a precipitation of the test materials was seen after 4 h exposure at dose 1.54 and above. Overall, the test item was considered to be non-mutagenetic under the conditions of the test.