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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Disperse Red 302 was found to be positive in the Ames test in bacteria. No other genotoxicity studies are not available on the test substance. Therefore, read-across from a similar substance is performed. It has been proposed to conduct in vivo Mammalian Erythrocyte Micronucleus test and Comet assay.

Link to relevant study records

Reference

Reference 1

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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

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

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.

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)

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.

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

 

	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

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-06-23 to 2014-09-25

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany)

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material:
20140120-2 (China)
- Expiration date of the lot/batch:
25 February 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
at room temperature
Physical State: solid
Colour: red

Target gene:

hypoxanthine-guanine-phosphoribosyl-transferase (HPRT)

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
- Periodically "cleansed" against high spontaneous background: 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 for experiment I (with and without metabolic activation):
0.0025, 0.0050, 0.010, 0.020, 0.030, 0.040, 0.050, 0.060, 0.070, 0.080, 0.090, 0.100, 0.125, 0.15 mM
Experiment I
with and without metabolic activation: 0.0010, 0.0025, 0.0050, 0.010, 0.020, 0.040, 0.060, 0.080, 0.100, 0.125 mM
Experiment II
without metabolic activation: 0.0010, 0.0025, 0.0050, 0.010, 0.020, 0.040, 0.060, 0.080, 0.100, 0.125 mM
and with metabolic activation: 0.0030, 0.0075, 0.015, 0.030, 0.045, 0.060, 0.075, 0.090, 0.011, 0.125 mM
Experiment III:
without metabolic activation: 0.07, 0.08, 0.09 mM

Vehicle / solvent:

Vehicle (Solvent) used: The test item was dissolved in DMSO and diluted in cell culture medium (MEM + 0 % FBS 4h treatment; MEM + 10 % FBS 20 h treatment) prior to treatment.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation; 300 µg/mL

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation; 0.8 and 1.0 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: dissolved in DMSO
DURATION: 4 h (short-term exposure), 20 h (long-term exposure)
Expression time (cells in growth medium): 5 days
Selection time (if incubation with selection agent): about one week

SELECTION AGENT (mutation assay) 11 µg/mL 6-thioguanine (TG)
NUMBER OF REPLICATIONS: two separate experiments (I+II) with single exposure; 5 individual flasks were seeded and evaluated
NUMBER OF CELLS EVALUATED: 400000 cells per flask
DETERMINATION OF CYTOTOXICITY: Method: relative growth

Evaluation criteria:

A test is considered to be negative if there is no biologically relevant increase in the number of mutants.
There are several criteria for determining a positive result:
-a reproducible three times higher mutation frequency than the solvent control for at least one of the concentrations.
-a concentration related increase of the mutation frequency; such an evaluation may be considered also in the case that a three-fold increase of
the mutant frequency is not observed.
-if there is by chance a low spontaneous mutation rate in the corresponding negative and solvent controls a concentration related increase of the mutations within their range has to be discussed.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment I without S9: 0.010, 0.040 and ≥ 0.080 mM; Experiment II without S9: ≥ 0.040 mM; Experiment III without S9:≥ 0.07 mM

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

FAT 93504/B is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.

Executive summary:

An in vitro experiment was performed to assess the potential of FAT 93504/B to induce gene mutations by means of a HPRT (hypoxanthine-guanine-phosphoribosyl-transferase) assay using the Chinese Hamster V79 cell line. The study was carried out according to OECD 476 guideline. In this mammalian cell gene mutation assay (HPRT locus), V79 cells cultured in vitro were exposed to FAT 93504/B at concentrations of

- 0.0010, 0.0025, 0.0050, 0.010, 0.020, 0.040, 0.060, 0.080, 0.100, 0.125 mM (with and without metabolic activation, Experiment I)

- 0.0010, 0.0025, 0.0050, 0.010, 0.020, 0.040, 0.060, 0.080, 0.100, 0.125 mM (without metabolic activation, Experiment II)

- 0.0030, 0.0075, 0.015, 0.030, 0.045, 0.060, 0.075, 0.090, 0.011, 0.125 mM (with metabolic activation, Experiment II)

- 0.07, 0.08, 0.09 mM (without metabolic activation, Experiment III).

FAT 93504/B was tested up to cytotoxic concentrations. Biologically relevant growth inhibition was observed in experiment I, II and III without metabolic activation. In experiment I without metabolic activation, the relative growth was 48.6 % for the highest concentration (0.125 mM) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 0.125 mM with a relative growth of 84.4 %. In experiment II without metabolic activation, the relative growth was 25.5 % for the highest concentration (0.125 mM) evaluated. The highest concentration evaluated with metabolic activation was 0.125 mM with a relative growth of 88.9 %. In experiment III without metabolic activation, the relative growth was 29.9 % for the highest concentration (0.09 mM) evaluated. In experiment I without metabolic activation the highest mutation rate (compared to the solvent control values) of 1.91 was found at a concentration of 0.01 mM with a relative growth of 67.7 %. In experiment I with metabolic activation the highest mutation rate (compared to the solvent control values) of 1.43 was found at a concentration of 0.080 mM with a relative growth of 83.9 %. In experiment II without metabolic activation the highest mutation rate (compared to the solvent control values) of 3.34 was found at a concentration of 0.080 mM with a relative growth of 32.7 %. In experiment II with metabolic activation the highest mutation rate (compared to the solvent control values) of 2.28 was found at a concentration of 0.015 mM with a relative growth of 113.3 %. In experiment III without metabolic activation the highest mutation rate (compared to the solvent control values) of 1.22 was found at a concentration of 0.08 mM with a relative growth of 35.5 %. The positive controls did induce the appropriate response. There was no evidence of a concentration related positive response of induced mutant colonies over background. Hence, FAT 93504/B is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.

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 1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Only four tester strains tested; no tester strain to detect cross-linking mutagens was included

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

Mutation in the histidine operon.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-liver extract from Aroclor 1254 induced rats

Test concentrations with justification for top dose:

Five dose levels: 0.2, 2, 20, 200 and 2000 micrograms per petri dish.

Vehicle / solvent:

DMSO: Dimethylsulfoxyde.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: MNNG= N-methyl-N'-nitro-N-nitrosoguanidine

Remarks:

TA 1535 and TA 100 - without S-9 activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA 1537 - without S-9 activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: daunomycine

Remarks:

TA 98 - without S-9 activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-anthramine

Remarks:

TA 1525, TA 1537, TA 98 and TA 100 - with S-9 activation

Details on test system and experimental conditions:

A sample of FAT 36052/E (red dyestuff) was tested in Salmonella typhimurium strains TA 1535, 1537, TA 98 and TA 100 at five dose levels: 0.2, 2, 20, 200 and 2000 micrograms per petri dish.
The substance was dissolved in DMSO, and every concentration was tested in triplicate. The tubes were agitated using a vortex mixed and poured onto the minimal medium base. The plates were incubated for 48 hours at 37 °C in the dark. The colonies, the revertants to the wilde type, were counted manually or electronically using a Fisher Colony counter.

Evaluation criteria:

The criteria of mutagenicity used in this test are a doubling of the spontaneous reversion rate and a dose-effect relationship.

Species / strain:

E. coli WP2

Metabolic activation:

not specified

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The evidence for mutagenicity existed only in the presence of a liver microsomal enzyme preparation (S-9 mix) from male rate pretreated with Aroclor 1254 and over a concentration range of 20 to 2000 micrograms of product per Petri dish.

Conclusions:

FAT 36052/E is not mutagenic for S. typhimurium strains TA 1535, TA 98 and TA 100 in the presence and absence of metabolic activation, but was mutagenic for S. typhimurium strain TA 1537 in presence of S-9 mix.

Executive summary:

FAT 36052/E was tested with the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 at five dose levels: 0.2, 2, 20, 200 and 200 µg (or nl) per petri dish both in presence and absence of metabolic activation. The test substance was dissolved in DMSO and every concentration was tested in triplicate. Under this experimental conditions, and employing a doubling of the spontaneous reversion rate and dose-effect relationship as criteria of mutagenicity, the product FAT 36052/E was not mutagenic for S. typhimurium strains TA 1535, TA 98 and TA 100 in the presence and absence of metabolic activation, but was mutagenic for S. typhimurium strain TA 1537 in presence of S-9 mix. The evidence for mutagenicity existed only in the presence of a liver microsomal enzyme preparation (S-9 mix) from male rats pretreated with Aroclor 1254 and over a concentration range of 20 to 2000 µg of product per petri dish.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Testing proposal for in-vivo micronucleus test and Comet assay.

Endpoint conclusion

Endpoint conclusion:

no study available (further information necessary)

Additional information

InVitro Ames test

FAT 36052/E was tested with the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 at five dose levels: 0.2, 2, 20, 200 and 200 µg (or nl) per petri dish both in presence and absence of metabolic activation. The test substance was dissolved in DMSO and every concentration was tested in triplicate. Under this experimental conditions, and employing a doubling of the spontaneous reversion rate and dose-effect relationship as criteria of mutagenicity, the product FAT 36052/E was not mutagenic for S. typhimurium strains TA 1535, TA 98 and TA 100 in the presence and absence of metabolic activation, but was mutagenic for S. typhimurium strain TA 1537 in presence of S-9 mix. The evidence for mutagenicity existed only in the presence of a liver microsomal enzyme preparation (S-9 mix) from male rats pretreated with Aroclor 1254 and over a concentration range of 20 to 2000 µg of product per petri dish.

In Vitro HPRT Read-across:

Data on mammalian cell gene mutation assay was not available for the target substance Disperse Red 302. To fill the data gaps, read across approach is adapted using similar substance FAT 93504/B. Read-across is claimed basis of structural relationship of the target and the source chemicals. Read across substance have been investigated for gene mutation potential. An in vitro experiment was performed to assess the potential of FAT 93504/B to induce gene mutations by means of a HPRT (hypoxanthine-guanine-phosphoribosyl-transferase) assay using the Chinese Hamster V79 cell line. The study was carried out according to OECD 476 guideline. In this mammalian cell gene mutation assay (HPRT locus), V79 cells cultured in vitro were exposed to FAT 93504/B at concentrations of  

- 0.0010, 0.0025, 0.0050, 0.010, 0.020, 0.040, 0.060, 0.080, 0.100, 0.125 mM (with and without metabolic activation, Experiment I)

- 0.0010, 0.0025, 0.0050, 0.010, 0.020, 0.040, 0.060, 0.080, 0.100, 0.125 mM (without metabolic activation, Experiment II)

- 0.0030, 0.0075, 0.015, 0.030, 0.045, 0.060, 0.075, 0.090, 0.011, 0.125 mM (with metabolic activation, Experiment II)

- 0.07, 0.08, 0.09 mM (without metabolic activation, Experiment III).

FAT 93504/B was tested up to cytotoxic concentrations. Biologically relevant growth inhibition was observed in experiment I, II and III without metabolic activation. In experiment I without metabolic activation, the relative growth was 48.6 % for the highest concentration (0.125 mM) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 0.125 mM with a relative growth of 84.4 %. In experiment II without metabolic activation, the relative growth was 25.5 % for the highest concentration (0.125 mM) evaluated. The highest concentration evaluated with metabolic activation was 0.125 mM with a relative growth of 88.9 %. In experiment III without metabolic activation, the relative growth was 29.9 % for the highest concentration (0.09 mM) evaluated. In experiment I without metabolic activation the highest mutation rate (compared to the solvent control values) of 1.91 was found at a concentration of 0.01 mM with a relative growth of 67.7 %. In experiment I with metabolic activation the highest mutation rate (compared to the solvent control values) of 1.43 was found at a concentration of 0.080 mM with a relative growth of 83.9 %. In experiment II without metabolic activation the highest mutation rate (compared to the solvent control values) of 3.34 was found at a concentration of 0.080 mM with a relative growth of 32.7 %. In experiment II with metabolic activation the highest mutation rate (compared to the solvent control values) of 2.28 was found at a concentration of 0.015 mM with a relative growth of 113.3 %. In experiment III without metabolic activation the highest mutation rate (compared to the solvent control values) of 1.22 was found at a concentration of 0.08 mM with a relative growth of 35.5 %. The positive controls did induce the appropriate response. In conclusion, there was no evidence of a concentration related positive response of induced mutant colonies over background. Hence, based on the read across data generated with FAT 93504/B, the target substance Disperse Red 302 is also considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.

 

In Vitro CA Read-across

Thein 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 thein 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.

 

In Vivo Micronucleus Read-across:

Currently no study is available for FAT 93503 is available for assessment of InVivo mutagenicity. However, a similar substance FAT 93504 was evaluated for the “Mammalian Erythrocyte Micronucleus Test” as per OECD Guideline No. 474, adopted on 29 July 2016. This study used 6 groups of rats and each group consisted of 6 males. The animals designated as group G1 animals were administered with corn oil as vehicle. The animals designated as groups G2, G3 and G4 were administered 500, 1000 and 2000 mg/kg bw/day of FAT 93504, respectively. The animals in group G6 were administered 250 mg/kg bw/day of the positive control ethyl methanesulfonate (for comet assay) and the animals in group G6 were administered 100 mg/kg bw/day of the positive control cyclophosphamide monohydrate (for micronucleus test) for three consecutive days by oral route using gavage cannula. Approximately 3 hours after the last dosing, all rats were sacrificed by cervical dislocation. The femurs were collected from each animal of G1 to G4 and G6 groups for bone marrow collection. Bone marrow cells were obtained by cutting open the epiphyses of femur bone immediately following sacrifice. The slides of bone marrow cells were stained with May-Gruenwald and Giemsa stain and observed for incidences of micronucleated polychromatic erythrocytes (MNPCE). The average percentage of MNPCEs was 0.06 in males dosed with vehicle. The animals dosed with the test item at 500, 1000 and 2000 mg/kg bw/day, the average percentage of MNPCEs were 0.06, 0.07 and 0.08, respectively. There was no statistically significant increase in the percentage of MNPCEs (per 4000 PCEs scored) at the doses of 500, 1000 and 2000 mg/kg bw/day of test item, in comparison with the vehicle control. The positive control group (G6), cyclophosphamide monohydrate at 100 mg/kg bw/day exhibited statistically significant increase in the numbers of MNPCEs when compared to vehicle control and the average percentage of MNPCEs (per 4000 PCEs scored) in positive control was 0.79. This demonstrated the sensitivity of the test system towards positive controls and confirmed that the test conditions were adequate. There was no statistically significant variation in body weight for treated animals. The dose formulation samples were analysed for homogeneity and dose concentration by HPLC and the formulation results were within the acceptance criteria of ± 15 % recovery to the nominal concentration. Based on the results obtained under the conditions employed during this experiment, it is concluded that the test item, FAT 93504 is neither clastogenic nor aneugenic at and up to 2000 mg/kg bw/day.

 

In Vivo Comet assay Read-across:

Currently no study is available for FAT 93503 is available for assessment of In Vivo mutagenicity. However, a similar substance FAT 93504 was evaluated in the “In vivo Mammalian Alkaline Comet Assay” as per OECD Guideline No. 489, adopted on 29 July 2016. This study was conducted to determine if the test item, FAT 93504, caused an increase in DNA damage in cells from specific organs. The animals designated as group G1 animals were administered with corn oil as vehicle. The animals designated as groups G2, G3 and G4 were administered 500, 1000 and 2000 mg/kg bw/day of FAT 93504, respectively. The animals in group G5 were administered 250 mg/kg bw/day of the positive control ethyl methane sulfonate (for comet assay). Approximately 3 hours after the last dosing, all rats were sacrificed by cervical dislocation and the designated organs (liver, duodenum, glandular stomach and testicles) were collected for G1 to G5 groups. The collected tissues were processed, single cells were isolated, and slides were prepared. Slides were run through submarine-type electrophoresis and drained. Drained slides were stained with ethidium bromide and evaluated for % tailing of DNA, i.e. tail length in microns measured from the estimated edge of the head region closest to the anode. The average % tailing for DNA from male liver cells was 3.21, 3.70, 3.99 and 3.76 at 0, 500, 1000 and 2000 mg/kg bw/day respectively. The average % tailing for DNA from glandular stomach cells were 3.41, 4.01, 3.97 and 4.28, respectively and in duodenum 3.07, 4.16, 4.26 and 3.94 at 0, 500, 1000 and 2000 mg/kg bw/day, respectively. There was no dose-related or statistically significant increase in the % tailing of DNA from cells of any of the organs for any of the FAT 93504 treated groups when compared to the vehicle control group. The average % tail DNA observed in the liver, glandular stomach and duodenum of the positive control (at 250 mg/kg bw/day of ethyl methyl sulfonate) dosed animals were 8.39, 11.33 and 9.93, respectively. The positive control [G5], ethyl methane sulfonate at a dose of 250 mg/kg bw/day produced a statistically significant increase in % tailing of DNA in cells from all the organs which were assessed (liver, duodenum, and glandular stomach) when compared to the equivalent cells from organs of vehicle control animals [G1]. These data support the conclusion that the test conditions and sensitivity of the COMET assay for this test of FAT 93504 were fully adequate. There was no statistically significant variation in body weight for treated animals. There were no gross and histopathology changes noted. The dose formulation samples were analysed for homogeneity and dose concentration by HPLC and the formulation results were within the acceptance criteria of ± 15 % recovery to the nominal concentration. Based on the results, it is concluded that FAT 93504 did not lead to increased DNA damage at and up to 2000 mg/kg bw/day.

Justification for classification or non-classification

Based on the findings of the genetic toxicity studies, the test substance is not considered to be classified according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.