Registration Dossier

Administrative data

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

Referenceopen allclose all

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-tationa

Statistical Signifi-canceb

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.

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 TE is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.

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:
other: S. typhimurium TA 1535, TA 98 and 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 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.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: gene mutation
Type of information:
experimental study planned
Study period:
Depending on ECHA approval
Justification for type of information:
TESTING PROPOSAL ON VERTEBRATE ANIMALS
Hazard endpoint for which vertebrate testing was proposed: Genetic toxicity in vivo with the registered substance.

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out: Disperse Red 302:1 (EC# 248-882-8)

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- Available GLP studies: There are no available GLP studies on the substance or on read-across analogues suitable to fill the endpoint.

- Available non-GLP studies: There are no available non-GLP studies on the substance or on read-across analogues suitable to fill the endpoint.

- Historical human data: There is no historical human data on the substance or on read-across analogues suitable to fill the endpoint.

- (Q)SAR: (Q)SAR analysis is not sufficient to fill the endpoint. There are no adequate models to address this end point.

- In vitro methods: Already available, but further in vivo data needed. Available studies: 1. Study with methodolgy equivalent to OECD Guideline 471 (Bacterial Reverse Mutation Assay); 2. OECD Guideline 476 (In vitro Mammalian Cell Gene Mutation Tests using the HPRT and xprt genes); 3. OECD Guideline 473 (In vitro Mammalian Chromosomal Aberration Test)

- Weight of evidence: There is not sufficient data on the substance or read across analogues to be able to establish a weight of evidence argument.

- Grouping and read-across: There is not sufficient data on the substance or read-across analogues to be able to group or propose read-across.

- Substance-tailored exposure driven testing [if applicable]: Not applicable
- Approaches in addition to above [if applicable]: Not applicable
- Other reasons [if applicable]: Not applicable

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- Test proposal is fully in line with ECHA guidance document*, and can neither be replaced by in vitro testing nor by using other data from other substances.
* Chapter R.7a: Endpoint specific guidance Version 4.1 – October 2015

FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
- Details on study design / methodology proposed: OECD Guideline 489: In Vivo Mammalian Alkaline Comet Assay
Qualifier:
according to guideline
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Executive summary:

The Registrant proposes to conduct the following test using the indicated test method:

 

In Vivo Mammalian Alkaline Comet Assay:OECD Guideline 489

 

The Registrant has assessed genotoxicity of FAT 93504/B TE in three in vitro systems.

In Reverse Mutation Assay using Bacteria (Salmonella Typhimurium) FAT 36052/D exerted a very weak mutagenic effect in this test system.

 

In vitro Mammalian Chromosome Aberration Test, the test substance FAT 93504/B TE induced structural chromosome aberrations in the V79 Chinese hamster cell line in the absence and the presence of metabolic activation.

In vitro Mammalian Cell Gene Mutation Test (HPRT-Locus) in Chinese Hamster V79 Cells,FAT 93504/B TE and its metabolites did not show any mutagenic activity in this forward mutation system.

 

According to REACH Annex IX 8.4 Column 2, if there is a positive result in any of the in vitro genotoxicity studies in Annex VII or VIII and there are no results available from an in vivo study already, an appropriate in vivo somatic cell genotoxicity study shall be proposed by the Registrant. The information on this endpoint is not available for the registered substance but needs to be present in the technical dossier to meet the data requirements. Consequently, there is a data gap and it is necessary to generate the data for this endpoint.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study planned
Study period:
Depending on ECHA approval
Justification for type of information:
TESTING PROPOSAL ON VERTEBRATE ANIMALS
Hazard endpoint for which vertebrate testing was proposed: Genetic toxicity in vivo with the registered substance.

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out: Disperse Red 302:1 (EC# 248-882-8)

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- Available GLP studies: There are no available GLP studies on the substance or on read-across analogues suitable to fill the endpoint.

- Available non-GLP studies: There are no available non-GLP studies on the substance or on read-across analogues suitable to fill the endpoint.

- Historical human data: There is no historical human data on the substance or on read-across analogues suitable to fill the endpoint.

- (Q)SAR: (Q)SAR analysis is not sufficient to fill the endpoint. There are no adequate models to address this endpoint.

- In vitro methods: Already available, but further in vivo data needed. Available study: OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

- Weight of evidence: There is not sufficient data on the substance or read across analogues to be able to establish a weight of evidence argument.

- Grouping and read-across: There is not sufficient data on the substance or read-across analogues to be able to group or propose read-across.

- Substance-tailored exposure driven testing [if applicable]: Not applicable
- Approaches in addition to above [if applicable]: Not applicable
- Other reasons [if applicable]: Not applicable

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- Test proposal is fully in line with ECHA guidance document*, and can neither be replaced by in vitro testing nor by using other data from other substances.
* Chapter R.7a: Endpoint specific guidance Version 4.1 – October 2015

FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
- Details on study design / methodology proposed: OECD Guideline 474: Mammalian Erythrocyte Micronucleus Test
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Endpoint conclusion
Endpoint conclusion:
no study available (further information necessary)

Additional information

The mutagenicity studies were performed with the test substances FAT 36052/E, FAT 36052/D and FAT 93504/B TE. Substances FAT 36052/D and FAT 93504/B are covered under the read-across. Hence, data of FAT 36052/E, FAT 36052/D and FAT 93504/B can be used to cover this endpoint for FAT 93503. In Reverse Mutation Assay using Bacteria (Salmonella Typhimurium) FAT 36052/E and FAT 36052/D were mutagenic in strain TA 1537 in presence of metabolic activation. 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 absence and the presence of metabolic activation. In vitro Mammalian Cell Gene Mutation Test (HPRT-Locus) in Chinese Hamster V79 Cells, FAT 93504/B and its metabolites did not show any mutagenic activity in this forward mutation system. According to REACH Annex IX 8.4 Column 2, if there is a positive result in any of the in vitro genotoxicity studies in Annex VII or VIII and there are no results available from an in vivo study already, an appropriate in vivo somatic cell genotoxicity study shall be proposed by the registrant. The information on this endpoint is not available for the registered substance but needs to be present in the technical dossier to meet the data requirements. Consequently, there is a data gap and it is necessary to generate the data for this endpoint. Therefore, a proposal to conduct the following tests using the indicated test method has been made to ECHA for the analogue substance FAT 93504/B:

 

EU Method B.12 - In Vivo Mammalian Erythrocyte Micronucleus test or the equivalent OECD Guideline 474 – Mammalian Erythrocyte Micronucleus test. 

In Vivo Mammalian Alkaline Comet Assay:OECD Guideline 489.

 

Justification for classification or non-classification

Based on the absence of adverse effect in the in-vitro genetic toxicity studies, Disperse Red 302 is considered not to classify for genotoxicity in accordance to the EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008. However, a testing proposal to perform an in vivo micronucleus study and Comet assay are made. The final classification will be proposed after completion of these test. According to REACH Annex IX 8.4 Column 2, if there is a positive result in any of the in vitro genotoxicity studies in Annex VII or VIII and there are no results available from an in vivo study already, an appropriate in vivo somatic cell genotoxicity study shall be proposed by the registrant. Therefore, the registrant proposes to conduct the following tests using the indicated test method:

EU Method B.12 Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus test or the equivalent OECD Guideline 474 – Mammalian Erythrocyte Micronucleus test.

In Vivo Mammalian Alkaline Comet Assay: OECD Guideline 489.