2-[(1-amino-9,10-dihydro-4-hydroxy-9,10-dioxo-2-anthryl)oxy]ethyl ethyl carbonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: other: clastogenicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-07-07 to 2014-09-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Refer chapter 13 for detailed read across justification.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

2-[(1-amino-9,10-dihydro-4-hydroxy-9,10-dioxo-2-anthryl)oxy]ethyl phenyl carbonate

EC Number:

248-882-8

EC Name:

2-[(1-amino-9,10-dihydro-4-hydroxy-9,10-dioxo-2-anthryl)oxy]ethyl phenyl carbonate

Cas Number:

28173-59-3

Molecular formula:

C23H17NO7

IUPAC Name:

2-[(1-amino-4-hydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl)oxy]ethyl phenyl carbonate

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 of Wistar phenobarbital and ß-naphthoflavone-induced rat liver S9 mix

Test concentrations with justification for top dose:

Pre-experiment:
with and without metabolic activation: 0.001, 0.0025, 0.005, 0.01, 0.025, 0.05, 0.075, 0.1, 0.125, 0.25 and 0.5 mM

The following concentrations were evaluated for the microscopic analysis of chromosomal aberrations:
Main Experiment:
without metabolic activation: 0.03, 0.04 and 0.05 mM
with metabolic activation: 0.03, 0.05 and 0.075 mM

Vehicle / solvent:

-Vehicle (s)/solvent(s) used: DMSO and cell culture medium
-Justification for choice of solvent/vehicle: Based on the results of the solubility test dimethylsulfoxide (DMSO) was used as solvent (1% DMSO v/v final concentration). Different test item stock solutions were prepared and added to the samples. The solvent was compatible with the survival of the cells and the S9 activity.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

cell culture medium

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation (900 µg/mL)

Controls 2

Untreated negative controls:

yes

Remarks:

cell culture medium

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation (1.11 µg/mL)

Details on test system and experimental conditions:

TREATMENT TIME:
4 hours (Experiment I with and without metabolic activation)

FIXATION INTERVAL: 20 hours (Experiment I with and without metabolic activation)
NUMBER OF REPLICATIONS: 1 experiment
NUMBER OF CELLS SEEDED: 1 x 10E4 - 5 x 10E4 cells
NUMBER OF CULTURES: two cultures per concentration
NUMBER OF CELLS SCORED: 200 cells per concentration (100 cells per culture),except for concentrations 0.05 and 0.075 mM (with metabolic activation) 400 cells were evaluated (200 cells per culture).
DETERMINATION OF CYTOTOXICITY: Mitotic index, cell count

Evaluation criteria:

There are several criteria for determining a positive result:
- a clear and dose-related increase in the number of cells with aberrations,
- a biologically relevant response for at least one of the dose groups, which is higher than the laboratory negative control range (0.0 % - 4.0 % without and 0.0 % - 4.3 % with metabolic activation).

Statistics:

A statistical evaluation was used as an aid for interpretation of the results. Statistical significance at the 5 % level (p <0.05) was evaluated by the Fischer´s exact test. The p value was used as a limit in judging for significance levels in comparison with the corresponding negative/solvent control.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Any other information on results incl. tables

 

	Dose Group	Concentration [mM]	Relative Mitotic Index [%]	Relative Cell Count [%]	Mean % Aberrant Cells		Historical Laboratory Negative Control Range	Precipi-tation^a	Statistical Signifi-cance^b
					incl. Gaps	excl. Gaps
without metabolic activation; 4 h treatment, 20 h preparation interval	C	0	100	100	1.5	1.0	0.0 % - 4.0 % aberrant cells	-	-
	S	0	100	100	2.0	0.0		-	-
	4	0.03	96	76	4.5	2.0		-	-
	5	0.04	72	89	11.0	4.0		+	+
	6	0.05	65	82	4.5	3.5		+	+
	EMS	900 μg/mL	106	76	12.0	10.0		-	+
with metabolic activation; 4 h treatment, 20 h preparation interval	C	0	96	98	5.0	1.5	0.0 % - 4.3 % aberrant cells	-	-
	S	0	100	100	4.0	3.5		-	-
	4	0.03	71	92	7.0	2.0		-	-
	6	0.05	62	90	9.5	5.5		-	-
	7	0.075	71	98	9.0	5.3		+	-
	CPA	1.11 μg/mL	113	98	18.0	15.0		-	+

The mitotic index was determined in 1000 cells per culture of each test group.

The cell count was determined by a cell counter per culture for each test group.

The relative values of the mitotic index and cell count are related to the solvent controls.

C: Negative Control (Culture Medium)

S: Solvent Control (DMSO)

EMS: Ethylmethanesulfonate

CPA:  Cyclophosphamide

a: - without precipitation, + with precipitation

b: statistical significant increase compared to negative controls (Fisher’s exact test, p<0.05), + significant; - not significant

Applicant's summary and conclusion

Conclusions:

FAT 93504/B is considered to be clastogenic in this chromosome aberration test using V79 cells in the presence of metabolic activation.

Executive summary:

The in vitro Mammalian Chromosome Aberration Test was carried out with FAT 93504/B according to OECD guideline 473 to assess its potential to induce structural chromosome aberrations in Chinese hamster V79 cells. Independent experiments were carried out with and without the addition of liver S9 mix from rats (exogenous metabolic activation). The metaphases were prepared at approximately 20 h after start of treatment with the test item. The treatment interval was 4 h without and with metabolic activation in the main experiment. Duplicate cultures were treated at each concentration. 100 metaphases per culture were scored for structural chromosomal aberrations. Based on the results of the solubility test dimethylsulfoxide (DMSO) was used as solvent (1 % DMSO v/v final concentration). Different test item stock solutions were prepared and added to the samples. The following concentrations were evaluated for the microscopic analysis of chromosomal aberrations:

Main Experiment:

without metabolic activation: 0.03, 0.04 and 0.05 mM

with metabolic activation: 0.03, 0.05 and 0.075 mM

In the pre-experiment, precipitation of the test item was detected without and with metabolic activation at concentrations of 0.05 and 0.075 mM with the unaided eye. In the main experiment, precipitation of the test item was observed without and with metabolic activation at concentrations of 0.04/0.05 and 0.075 mM, respectively. In the main experiment without metabolic activation, no cytotoxic effects of the test item were observed at all evaluated concentrations considering the relative cell count. However, the relative mitotic index was decreased and the highest concentration of 0.05 mM showed cytotoxic effects (rel. mitotic index below 70 %). With metabolic activation no cytotoxic effects of the test item were observed at all concentrations considering the relative cell count. The relative mitotic index of the dose groups evaluated was decreased (0.03 mM (71 %), 0.04 mM (62 %), 0.05 mM (71 %)) which indicates a slight cytotoxicity of the test item (rel. mitotic index below 70 %). In the main experiment without metabolic activation no biologically relevant increase of aberrant cells was determined at all evaluated concentrations compared to the solvent control. With metabolic activation increases of aberrant cells were observed at concentrations of 0.05 mM and 0.075 mM compared to the solvent control. These increases of aberrant cells were considered as biologically relevant. In all experiments vehicle controls gave frequencies of aberrations within the range expected for the V79 cell line. Both positive controls, EMS (900 μg/mL) and CPA (1.11 μg/mL) induced distinct and biologically relevant increase in the number of cells containing structural chromosomal aberrations. According to the results of the present study, the test substance did lead to a biologically relevant increase in the number of cells with chromosome aberrations after adding a metabolizing system (S9 mix). Furthermore, an increase in the number of polyploid cells was observed at the highest test item concentration of 0.075 mM (with metabolic activation). Thus, under the experimental conditions described, with regard to the data obtained in the in vitro Mammalian Chromosome Aberration Test, the test substance FAT 93504/B induced structural chromosome aberrations in the V79 Chinese hamster cell line in the presence of metabolic activation. The positive controls induced the appropriate responses.