5,12-dihydroquino[2,3-b]acridine-7,14-dione

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Mammalian Cell Gene Mutation Tests

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 July 2021 to 04 November 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted on 29th July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: In vitro Mammalian Cell Gene Mutation Tests using the Hprt and xprt genes

Test material

Test material information

Constituent 1

Reference substance name:

5,12-dihydroquino[2,3-b]acridine-7,14-dione

EC Number:

213-879-2

EC Name:

5,12-dihydroquino[2,3-b]acridine-7,14-dione

Cas Number:

1047-16-1

Molecular formula:

C20H12N2O2

IUPAC Name:

5,7,12,14-tetrahydro-5,12-diazapentacene-7,14-dione

Test material form:

solid: nanoform

Method

Target gene:

CHO AA8 cells

Species / strain

Species / strain / cell type:

other: CHO AA8 cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: American Type Culture Collection (ATCC)
- Cell doubling time : Approximately 12 hours

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Alpha MEM CULTURE MEDIA with 10% FBS and 1% penstrep and 5±1% CO2
- Properly maintained: [yes]
- Periodically checked for Mycoplasma contamination: [yes]
- Doubling time model chromosome number cells were checked.

Metabolic activation:

with and without

Metabolic activation system:

S9 homogenate

Test concentrations with justification for top dose:

15.625, 31.25, 62.5 and 125 µg/mL based on initial cytotoxicity test

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 2×107

DURATION
- Exposure duration: 3 hours and 38 minutes
- Expression time (cells in growth medium): 7 days for mutant phenotype expression
- Selection time (if incubation with a selection agent):8 days

SELECTION AGENT (mutation assays): 10 µM of 6-Thioguanine

STAIN : 5% giemsa

NUMBER OF REPLICATIONS: 5

METHODS STAINING TECHNIQUE USED: Post incubation period, medium from each culture flask was aspirated and stained with 5% Giemsa stain. Number of colonies formed was counted manually.


DETERMINATION OF CYTOTOXICITY
- Method:cloning efficiency

Rationale for test conditions:

Acceptance of a test is based on the following criteria:
•The concurrent vehicle control is considered acceptable for addition to the laboratory historical vehicle control database as described in OECD guidelines for testing of chemicals, No. 476.
•Concurrent positive controls should induce responses that are compatible with those generated in the historical positive control data base and produce a statistically significant increase compared with the concurrent negative/vehicle control.
•Two experimental conditions (i.e. with and without metabolic activation) were tested unless one resulted in positive results.
•Adequate number of cells and concentrations are analysable (according to OECD guidelines for testing of chemicals, No. 476).
•The criteria for the selection of top concentration are consistent with those described in OECD guidelines for testing of chemicals, No. 476.

Evaluation criteria:

Colony counts in selective media and non-selective media

Statistics:

yes, Statistical analysis of the experimental data was carried out using SPSS Statistical package version 22.0.

Results and discussion

Test results

Key result

Species / strain:

other: CHO AA8 cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No change in pH was observed in any of the test concentrations
- Water solubility: Insoluble
- Precipitation: The precipitation and pH were tested at 0.03125, 0.0625, 0.125, 0.25, 0.5, 1 and 2 mg/mL concentrations. Post 3 hours of incubation, no precipitation was observed at the tested concentrations of 0.03125, 0.0625 mg/mL, moderate precipitation was observed at 0.125 and 0.25 mg/mL, heavy precipitation was observed at 0.5, 1 and 2 mg/mL

RANGE-FINDING/SCREENING STUDIES: Yes

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
Positive Control- Benzo(a)pyrene and 4- Nitroquinoline N-oxide
With Metabolic Activation
(3 to 6 hours)
[Benzo(a)pyrene] Without Metabolic Activation
(3 to 6 hours)
[4 Nitroquinoline N-oxide]
Mean Data of Mutant Frequency/2x106 Cells 261.94 264.60
Standard
Deviation 27.28 18.52
Margin of Error 17.82 12.10
Upper bound 279.76 276.70
Lower bound 244.12 252.50

- Negative (solvent/vehicle) historical control data: Negative controls were maintained.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Relative survivality

Any other information on results incl. tables

 

TABLE 1.          SUMMARY OF INITIAL CYTOTOXICITY TEST

Set No.	Treatment	Concentration (µg/mL)	Average Colony Count± SD			Cloning           Efficiency (CE)	Adjusted Cloning Efficiency (ACE)	Relative Survival (RS) (%)
Set 1 +S9	Vehicle Control  (Acetone)	-	189.67	±	2.08	0.95	1.15	-
	test item	7.8125	185.67	±	1.53	0.93	1.10	95.65
		15.625	180.67	±	5.03	0.90	1.06	92.17
		31.25	175.33	±	3.51	0.88	1.00	86.96
		62.5	164.33	±	4.04	0.82	0.88	76.52
		125	144.00	±	5.20	0.72	0.72	62.61
Set 2 -S9	Vehicle Control  (Acetone)	-	187.67	±	2.52	0.94	1.13	-
	test item	7.8125	182.33	±	2.52	0.91	1.06	93.81
		15.625	182.00	±	3.00	0.91	1.04	92.04
		31.25	178.00	±	2.65	0.89	0.99	87.61
		62.5	165.00	±	4.58	0.83	0.90	79.65
		125	142.67	±	9.45	0.71	0.73	64.60

 +S9: with metabolic activation; -S9: without metabolic activation;

 Adjusted CE = CE × Number of cells at the end of treatment/number of cells at the beginning of treatment.

 RS = Adjusted CE in treated culture/Adjusted CE in the vehicle control × 100.

 CE = Number of colonies/Number of cells plated.

 

 

TABLE 1.           SUMMARY OF PARALLEL CYTOTOXICITY TEST- GENE MUTATION TEST

Set No.	Treatment	Concentration (µg/mL)	Average Colony Count ± SD			Cloning Efficiency (CE)	Adjusted Cloning Efficiency (ACE)	Relative Survival (RS) (%)
Set 1 +S9	Vehicle Control (Acetone)	-	190.33	±	1.53	0.95	1.17	-
	Negative control	-	190.67	±	1.15	0.95	1.17	100.00
	test item	15.625	182.00	±	2.00	0.91	1.09	93.16
		31.25	172.67	±	4.51	0.86	0.99	84.62
		62.5	168.33	±	2.08	0.84	0.90	76.92
		125	147.67	±	2.52	0.74	0.75	64.10
	Benzo(a)pyrene              (Positive Control)	3	180.00	±	7.81	0.90	1.07	91.45
Set 2 -S9	Vehicle Control (Acetone)	-	188.67	±	5.03	0.94	1.15	-
	Negative control	-	189.33	±	2.08	0.95	1.19	103.48
	test item	15.625	178.67	±	1.15	0.89	1.07	93.04
		31.25	174.33	±	5.03	0.87	1.01	87.83
		62.5	167.33	±	5.51	0.84	0.88	76.52
		125	143.00	±	4.58	0.72	0.73	63.48
	4 Nitroquinoline N-oxide (Positive Control)	1	179.33	±	4.51	0.90	1.09	94.78

 +S9: with metabolic activation;  -S9: without metabolic activation;   

 *Note: Cloning Efficiency = 200 cells plated for each replicate.

 RS = Adjusted CE in treated culture/Adjusted CE in the vehicle control × 100.

 CE = Number of colonies/Number of cells plated.

 Adjusted CE = CE × Number of cells at the end of treatment/number of cells at the beginning of treatment.

 

 

Set No.	Treatment	Concentration (µg/mL)	*Average Colony Count ± SD			Cloning Efficiency in selective media	Cloning Efficiency in non-selective media*	Total number of Mutant Colonies/ 2×10^6cells	Mutant Frequency/ 2×10^6cells
Set 1 +S9	Vehicle Control (Acetone)	-	186.67	±	4.04	0.0000105	0.93	21	22.58
	Negative control	-	187.00	±	2.65	0.0000110	0.94	22	23.40
		15.625	186.00	±	1.00	0.0000110	0.93	22	23.66
		31.25	183.33	±	3.79	0.0000110	0.92	22	23.91
		62.5	178.67	±	2.31	0.0000110	0.89	22	24.72
		125	175.67	±	3.51	0.0000105	0.88	21	23.86
	Benzo(a)pyrene              (Positive Control)	3	181.67	±	1.53	0.0001140	0.91	228	250.55**
Set 2 -S9	Vehicle Control (Acetone)	-	188.33	±	2.08	0.0000115	0.94	23	24.47
	Negative control	-	187.33	±	4.04	0.0000115	0.94	23	24.47
	test item	15.625	184.67	±	5.51	0.0000115	0.92	23	25.00
		31.25	183.00	±	5.57	0.0000120	0.92	24	26.09
		62.5	182.33	±	6.03	0.0000110	0.91	22	24.18
		125	174.33	±	5.03	0.0000115	0.87	23	26.44
	4 Nitroquinoline N-oxide (Positive Control)	1	183.33	±	4.51	0.0001170	0.92	234	254.35**

TABLE 1.           SUMMARY OF GENE MUTATION TEST

 

+S9: with metabolic activation; -S9: without metabolic activation.                                                                                                  

 *Note: Cloning efficiency = 200 cells plated for each replicate.  

 **: Statistically significant (p˂0.05). 

Mutant Frequency = Cloning efficiency of mutant colonies in selective medium/Cloning efficiency in non- selective medium.

Applicant's summary and conclusion

Conclusions:

The test item is considered as non-mutagenic at and up to the concentration of 125 µg/mL, both in the presence and absence of metabolic activation under the tested laboratory conditions.

Executive summary:

The test item was evaluated for gene mutation test in CHO AA8 cells.

The test item formed a suspension in acetone at concentration of 200 mg/mL. After 3 hours of incubation, no precipitation was observed at the tested concentrations of 0.03125 and 0.0625 mg/mL, moderate precipitation was observed at 0.125 and 0.25 mg/mL, and heavy precipitation was observed at 0.5, 1 and 2 mg/mL. No change in pH was observed in any of the test concentrations.

On the basis of precipitation results, 0.125 mg/mL (125 µg/mL) was selected as the highest concentration for the initial cytotoxicity test. Initial cytotoxicity test was conducted at the concentrations of 7.8125, 15.625, 31.25, 62.5 and 125 µg/mL using acetone as a vehicle in four flasks/group in the presence and absence of metabolic activation (4 hours and 5 minutes). Cytotoxicity was assessed by determining the Adjusted Cloning Efficiency and Relative Survival in the test.

The results of the initial cytotoxicity test indicated that the Relative Survival was greater  than 10% (62.61 % in presence of metabolic activation and64.60 % in absence of metabolic activation) at 125 µg/mL when compared with the respective vehicle control, both in the presence and absence of metabolic activation. Based on these results,         125 µg/mL was selected as highest concentration for gene mutation test.

The gene mutation test was conducted at the concentrations of 125, 62.5, 31.25 and     15.625 µg/mL using acetone as a vehicle in four plates/group in the presence and absence of metabolic activation (3 hours and 38 minutes).Benzo(a) pyrene and4 Nitroquinoline N-oxidewere used aspositive controlsfor the gene mutation test.

Cytotoxicity as Relative Survival was 64.10 % in presence of metabolic activation and63.48 % in absence of metabolic activationat the highest tested concentration of 125µg/mL. Relative Survival of negative control was 100.00 in presence of metabolic activation and 103.48 in absence of metabolic activation.There was no statistically significant increase in mutant frequencies at any of the concentrations tested and negative control when compared with the vehicle control.

There was statistically significant increase in mutant frequenciesfor positive controlswhen compared with the vehicle controlin bothmetabolic activation andabsence of metabolic activation.