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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance was found to be weak mutagenic in Ames test. It also induced structural chromosome aberration in Chinese hamster cell line. In HPRT locus assay, the test substance did not showed any mutagenic activity in forward mutation system.

Link to relevant study records

Referenceopen allclose all

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
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 20h 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
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:
not examined
Positive controls validity:
valid

None

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

In a 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 responseof 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.

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 - 2014-09-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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:
not examined
Positive controls validity:
valid

 

Dose Group

Concentration [mM]

Relative Mitotic Index [%]

Relative Cell Count
[%]

Mean %

Aberrant Cells

Historical Laboratory Negative Control Range

Precipitationa

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

-

+

 

 

withmetabolic 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 and the substance FAT 93504/B was assessed for 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 bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 1982
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
conducted according to previous version available in 1981.
Deviations:
yes
Remarks:
Only four tester strains were used for the experiment and no tester strain to detect cross-linking mutagens was included.
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
The 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 µg per petri dish.
Vehicle / solvent:
DMSO: Dimethylsulfoxide.
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
Details on test system and experimental conditions:
A sample of FAT 36052/D (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 µg 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 wild 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-response relationship.
Species / strain:
E. coli WP2
Metabolic activation:
not specified
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not specified
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
not specified
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 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
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 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 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
Additional information on results:
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.

None

Conclusions:
FAT 36052/D 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:

The substance FAT 36052/D 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 microgramme (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 the experimental conditions, and employing a doubling of the spontaneous reversion rate and dose-effect relationship as criteria of mutagenicity, the product FAT 36052/D 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 microgramme of product per petri dish.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

FAT 93504 was found to be non-mutagenic and non-clastogenic in invivo studies.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Study initiation date : 20 December 2021
Experimental starting date : 23 December 2021
Experimental completion date : 15 April 2022
Study completion date - 15 July 2022
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian erythrocyte micronucleus test
Specific details on test material used for the study:
Name of Test Item: FAT 93504
Physical Appearance (with colour): Solid (Red)
Batch No.: B/TE (20140120-2 (china))
Purity (as per certificate of analysis): 88.5 %
Storage Conditions: Ambient (21 to 29 ºC)
Test Item Code by Test Facility: D1303-001
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat is one of the recommented species by regulatory agencies for conducting in vivo mammalian erythrocyte micronucleus test
Sex:
male
Details on test animals or test system and environmental conditions:
Source of supply: In-house bred animals.
Animals were housed under standard laboratory conditions, in an environmentally monitored air-conditioned room with adequate fresh air supply (12 to 15 air changes per hour), room temperature 19.7 to 22.8 ºC and relative humidity 45 % to 65 % in main study, with 12 hours fluorescent light and 12 hours dark cycle. The temperature and relative humidity were recorded once daily.
Route of administration:
oral: gavage
Vehicle:
Corn oil
Details on exposure:
The test item was administered through oral route once a day for 3 consecutive days (0 day, 24 hours and 45 hours), using gavage cannula. All the doses were administered in an equal volume of 10 mL/kg bw/day the dose of 500 (G2), 1000 (G3) and 2000 (G4) mg/kg bw/day as low, mid and high dose, respectively. Vehicle control group (G1) animals were administered with vehicle. Positive control group (G6) animals were administered with cyclophosphamide monohydrate, at the dose volume of 10 mL/kg bw/day. Ethyl methanesulfonate and cyclophosphamide monohydrate were dissolved in distilled water and administered at a dose of 250 and 100 mg/kg bw/day, respectively.
Duration of treatment / exposure:
3 days
Frequency of treatment:
Once in every day
Post exposure period:
Animals were euthanized by cervical dislocation 3 hours after the third treatment and subjected to gross pathological examination. Bone marrow was collected for micronucleus test.
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Vehicle control
Dose / conc.:
500 mg/kg bw/day
Remarks:
Low dose group
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
Mid dose group
Dose / conc.:
2 000 mg/kg bw/day
Remarks:
High dose group
No. of animals per sex per dose:
6 animals per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
Ethyl methanesulfonate
Tissues and cell types examined:
Bone marrow cells
Details of tissue and slide preparation:
SAMPLING TIME AND TISSUE COLLECTION
Animals were euthanized by cervical dislocation 3 hours after the third treatment and subjected to gross pathological examination. Bone marrow was collected for micronucleus test.
The femurs were isolated from each animal for bone marrow collection (except from G5 group). Bone marrow cells were obtained by cut opening the epiphyses of femur bone immediately following sacrifice. The marrow was flushed out into a centrifuge tube using the Foetal Bovine Serum (FBS). The femur bone marrow cells were centrifuged at about 2700±100 rpm for 10 minutes. Prior to smear preparation, the supernatant were discarded and the cell pellet was then resuspended in approximately 50 µL of Foetal Bovine Serum (FBS).
Evaluation criteria:
BONE MARROW SMEARS SLIDE EVALUATION
All the slides including those of positive and vehicle controls were coded before microscopic evaluation to avoid group bias during evaluation. For each animal, a minimum of 500 erythrocytes (which included mature and immature erythrocytes) were scored from first slide of the animal to determine PCE: total RBC ratio along with the incidence of micronucleus. The subsequent slides were scored only for the number of PCEs and incidence of micronucleated PCEs. For each animal, a minimum of 4000 polychromatic erythrocytes (PCEs) were scored for the incidence of micronucleated immature erythrocytes (MNPCEs).
Statistics:
Body weight of day 1, 2 and 3 was analyzed by SPSS version no. 27, at a 95 % level (p ≤0.05) of significance. Intergroup comparison of body weight of Day 1, 2 and 3 were done. Slides from main study were decoded after analysis, the number of PCE (polychromatic erythrocytes), RBC (red blood corpuscles), MNPCE (micronucleated polychromatic erythrocytes) and PCE/total erythrocytes ratio (polychromatic erythrocytes/ total erythrocytes) and frequency of MNPCE was calculated. The data of positive control and the treatment groups were compared with that of the vehicle control for the incidence of MNPCEs and the proportion of PCEs among total RBCs by SPSS at a 95 % level (p ≤0.05) of significance. All analysis and comparisons were evaluated at the 95 % level of confidence (p <0.05). Percentage of micronucleus comparison between treated and control groups. Statistically significant changes obtained were designated by the superscripts in the summary table throughout the report as stated below:

*: Statistically significant (p <0.05).
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
CLINICAL SIGNS OF TOXICITY AND MORTALITY
The animals dosed with the test item resulted in no clinical signs or mortality in any of the treated animals.

BODY WEIGHT
No statistically significant changes in body weight were observed in any of the treated animals when compared to vehicle control group.

GROSS PATHOLOGY
No gross pathological findings were observed in any of the animals dosed with the test item at the doses of 500, 1000 and 2000 mg/kg bw/day.

PCE: Total RBC Ratio and Incidence of Micronucleus
The average numbers of micronucleated polychromatic erythrocytes (MNPCE) were observed for a minimum of 4000 polychromatic erythrocytes (PCEs). The incidence of percent MNPCEs were 0.06, 0.07 and 0.08 in males treated with FAT 93504 at 500, 1000 and 2000 mg/kg bw/day respectively in comparison with the vehicle dosed animals.
In the positive control animals, the incidence of percent MNPCEs was 0.79.

TABLE 1 INDIVIDUAL ANIMAL CLINICAL SIGNS AND MORTALITY






















































































































































































































































Sex



Group & Dose (mg/kg)



Animal No.



Day



1



2



3



am



pm



am



pm



am



am



Male


 



G1 & 0



Rg5006



N



N



N



N



N



N



Rg5007



N



N



N



N



N



N



Rg5008



N



N



N



N



N



N



Rg5009



N



N



N



N



N



N



Rg5010



N



N



N



N



N



N



Rg5011



N



N



N



N



N



N



G2 & 500



Rg5012



N



N



N



N



N



N



Rg5013



N



N



N



N



N



N



Rg5014



N



N



N



N



N



N



Rg5015



N



N



N



N



N



N



Rg5016



N



N



N



N



N



N



Rg5017



N



N



N



N



N



N



G3 & 1000



Rg5018



N



N



N



N



N



N



Rg5019



N



N



N



N



N



N



Rg5020



N



N



N



N



N



N



Rg5021



N



N



N



N



N



N



Rg5022



N



N



N



N



N



N



Rg5023



N



N



N



N



N



N



G4 & 2000



Rg5024



N



N



N



N



N



N



Rg5025



N



N



N



N



N



N



Rg5026



N



N



N



N



N



N



Rg5027



N



N



N



N



N



N



Rg5028



N



N



N



N



N



N



Rg5029



N



N



N



N



N



N



N: Normal Normal, EMS: Ethyl methanesulfonate


TABLE 1. (Contd.…). INDIVIDUAL ANIMAL CLINICAL SIGNS AND MORTALITY

















































































Sex



Group & Dose (mg/kg)



Animal No.



Day



1



2



3



am



pm



am



pm



am



am



Male


 



G6 & 100


(CPA)



Rg5036



N



N



N



N



N



N



Rg5037



N



N



N



N



N



N



Rg5038



N



N



N



N



N



N



Rg5039



N



N



N



N



N



N



Rg5040



N



N



N



N



N



N



Rg5041



N



N



N



N



N



N



N: Normal, CPA: Cyclophosphamide monohydrate.


TABLE 2 INDIVIDUAL ANIMAL BODY WEIGHT



















































































































































































































Sex



Group & Dose (mg/kg)



Animal No.



Day 1



Day 2



Day 3



Male



G1 & 0



Rg5006



209.60



212.97



218.60



Rg5007



232.39



238.11



242.90



Rg5008



257.86



260.02



267.21



Rg5009



248.14



250.06



259.22



Rg5010



255.49



258.97



268.02



Rg5011



271.01



278.58



284.90



Mean



245.75



249.79



256.81



±SD



21.77



22.41



23.16



G2 & 500



Rg5012



212.11



229.86



220.70



Rg5013



220.40



217.65



211.60



Rg5014



250.66



253.11



250.17



Rg5015



256.15



259.13



255.15



Rg5016



261.81



259.96



259.01



Rg5017



275.82



278.3



274.19



Mean



246.16



249.67



245.14



±SD



24.77



22.11



24.02



G3 & 1000



Rg5018



212.48



219.56



210.46



Rg5019



223.09



220.13



212.31



Rg5020



240.64



245.69



240.10



Rg5021



253.65



252.18



245.11



Rg5022



264.54



265.51



259.01



Rg5023



278.69



281.65



271.75



Mean



245.52



247.45



239.79



±SD



25.09



24.68



24.64



G4 & 2000



Rg5024



220.29



218.90



211.90



Rg5025



225.71



227.99



221.11



Rg5026



248.30



248.61



242.12



Rg5027



251.88



253.00



249.01



Rg5028



263.96



261.69



254.75



Rg5029



275.39



275.58



268.18



Mean



247.59



247.63



241.18



±SD



21.37



21.07



21.14



SD: Standard deviation


TABLE 2. (Contd.…). INDIVIDUAL ANIMAL BODY WEIGHT (g)          
































































Sex



Group & Dose (mg/kg)



Animal No.



Day 1



Day 2



Day 3



Male



G6 & 100


(CPA)



Rg5036



219.06



222.10



220.18



Rg5037



235.79



236.97



234.18



Rg5038



255.94



254.06



253.11



Rg5039



256.10



257.58



255.18



Rg5040



263.59



266.77



259.80



Rg5041



263.26



265.06



261.18



Mean



248.96



250.42



247.24



±SD



17.81



17.49



16.50



SD: Standard deviation, CPA: Cyclophosphamide monohydrate.


TABLE 3 INDIVIDUAL ANIMAL GROSS PATHOLOGY 

















































































































     Sex



Group & Dose


(mg/kg)



Animal No.



Gross Pathology Findings


(Internal/External)



Males


 



G1 & 0



Rg5006



NAD



Rg5007



NAD



Rg5008



NAD



Rg5009



NAD



Rg5010



NAD



Rg5011



NAD



G2 & 500



Rg5012



NAD



Rg5013



NAD



Rg5014



NAD



Rg5015



NAD



Rg5016



NAD



Rg5017



NAD



G3 & 1000



Rg5018



NAD



Rg5019



NAD



Rg5020



NAD



Rg5021



NAD



Rg5022



NAD



Rg5023



NAD



G4 & 2000



Rg5024



NAD



Rg5025



NAD



Rg5026



NAD



Rg5027



NAD



Rg5028



NAD



Rg5029



NAD



  NAD: No Abnormalities Detected


TABLE 3. (Contd.…). INDIVIDUAL ANIMAL GROSS PATHOLOGY






































Sex



Group & Dose


(mg/kg)



Animal No.



Gross Pathology Findings


(Internal/External)



Males


 



G6 & 100


(CPA)



Rg5036



NAD



Rg5037



NAD



Rg5038



NAD



Rg5039



NAD



Rg5040



NAD



Rg5041



NAD



NAD: No Abnormalities Detected, CPA: Cyclophosphamide monohydrate.


TABLE 4 INDIVIDUAL ANIMAL MICRONUCLEUS DATA











































































































































































































Sex



Group & Dose


(mg /kg)



Animal No.



Total NCEs



Total PCEs



PCE: Total Erythrocytes Ratio



Mean of PCE: Total Erythrocytes Ratio



+SD



% Reduction of


PCE: Total Erythrocytes


Ratio



Total No. of PCE's Scored



Total Number of MNPCEs



% of MNPCEs



Mean of % of MNPCEs



Males



G1 & 0


 



Rg5006



242



260



0.52



0.51



0.01



NA



4050



3



0.07



0.06



Rg5007



250



252



0.50



4033



2



0.05



Rg5008



248



255



0.51



4059



2



0.05



Rg5009



253



259



0.51



4028



2



0.05



Rg5010



249



258



0.51



4077



3



0.07



 



Rg5011



250



253



0.50



4047



3



0.07



G2 & 500



Rg5012



259



251



0.49



0.50



0.01



1.96



4043



2



0.05



0.06



Rg5013



269



253



0.49



4080



2



0.05



Rg5014



255



247



0.49



4013



3



0.07



Rg5015



250



250



0.50



4066



3



0.07



Rg5016



248



257



0.51



4057



2



0.05



 



Rg5017



253



248



0.50



4077



2



0.05



G3 & 1000



Rg5018



270



250



0.48



0.48



0.00



5.88



4042



2



0.05



0.07



Rg5019



277



252



0.48



4050



3



0.07



Rg5020



268



248



0.48



4039



2



0.05



Rg5021



278



247



0.47



4059



4



0.10



Rg5022



271



252



0.48



4069



4



0.10



 



 



Rg5023



273



253



0.48



4027



3



0.07



   SD: Standard deviation.                                                                              


TABLE 4. (Contd.…). INDIVIDUAL ANIMAL MICRONUCLEUS DATA














































































































































Sex



Group & Dose


(mg /kg)



Animal No.



Total NCEs



Total PCEs



PCE: Total Erythrocytes Ratio



Mean of PCE: Total Erythrocytes Ratio



+SD



% Reduction of


PCE: Total Erythrocytes


Ratio



Total No. of PCE's Scored



Total Number of MNPCEs



% of MNPCEs



Mean of % of MNPCEs



Males



G4 & 2000


 



Rg5024



282



238



0.46



0.47



0.01



7.84



4053



1



0.02



0.08



Rg5025



284



240



0.46



4049



4



0.10



Rg5026



289



254



0.47



4032



3



0.07



Rg5027



275



246



0.47



4072



4



0.10



Rg5028



279



253



0.48



4090



4



0.10



 



Rg5029



274



244



0.47



4028



3



0.07



G6 & 100 CPA



Rg5036



289



243



0.46



0.46



0.01



9.80



4058



31



0.76



0.79*



Rg5037



275



240



0.47



4087



30



0.73



Rg5038



282



237



0.46



4053



34



0.84



Rg5039



285



244



0.46



4044



32



0.79



Rg5040



289



251



0.46



4064



32



0.79



 



Rg5041



293



243



0.45



4070



33



0.81



 SD: Standard deviation, CPA: Cyclophosphamide monohydrate, *: Statistically significant


 

Conclusions:
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.
Executive summary:

The test item 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.

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Study initiation date : 20 December 2021 Experimental starting date : 23 December 2021 Experimental completion date : 25 March 2022 Study Completion date : 15 July 2022
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
This study was conducted to determine if the test item, FAT 93504, caused an increase in DNA damage in cells from specific organs. The comet assay can detects single and double stranded breaks when DNA is analyzed under alkaline conditions (≥pH 13). These strand breaks, when they occur in vivo, may be repaired, resulting in no persistent effect, or may be lethal to the cell, or may be fixed into a mutation resulting in a permanent viable change.
Qualifier:
according to guideline
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Version / remarks:
Adopted on 29th July 2016
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian comet assay
Specific details on test material used for the study:
Name of Test Item: FAT 93504
Physical Appearance (with colour): Solid (Red)
Batch No.: B/TE (20140120-2 (china))
Purity (as per certificate of analysis): 88.5 %
Storage Conditions: Ambient (21 to 29 ºC)
Test Item Code by Test Facility: D1303-001
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Rat is one of the recommended species by regulatory agencies for conducting in vivo comet assay among rodents.
Sex:
male
Details on test animals or test system and environmental conditions:
Animals were housed under standard laboratory conditions, in an environmentally monitored air-conditioned room with adequate fresh air supply (12 to 15 air changes per hour), room temperature 19.7 to 22.8 ºC and relative humidity 45 % to 65 % , with 12 hours fluorescent light and 12 hours dark cycle.
Route of administration:
oral: gavage
Vehicle:
Corn oil
Details on exposure:
The test item was administered through oral route once a day for 3 consecutive days (0 day, 24 hours and 45 hours) using gavage cannula. All the doses were administered in an equal volume of 10 mL/kg bw/day, the dose of 500 (G2), 1000 (G3) and 2000 (G4) mg/kg bw/day as low, mid and high dose, respectively. Vehicle control group (G1) animals were administered with corn oil. Positive control group G5 animals were administered with ethyl methanesulfonate at the dose volume of 10 mL/kg bw/day.
Duration of treatment / exposure:
3 days
Frequency of treatment:
Once in a day
Post exposure period:
Animals were euthanized by cervical dislocation 3 hours after the third treatment and subjected to gross pathological examination. Liver, glandular stomach, duodenum and testicles were collected for the comet assay.
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1 : Vehicle control
Dose / conc.:
500 mg/kg bw/day
Remarks:
Group 2 : Low dose group
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
Group 3 : Mid dose group
Dose / conc.:
2 000 mg/kg bw/day
Remarks:
Group 4 : High dose group
No. of animals per sex per dose:
6 males per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
Ethyl methanesulfonate
Tissues and cell types examined:
Liver, glandular stomach and duodenum cells were analysed for comet assay parameters and testes cells were not analysed for comet assay parameters
Details of tissue and slide preparation:
TISSUE COLLECTION
Animals were euthanized by cervical dislocation 3 hours after the third treatment and subjected to gross pathological examination. Liver, glandular stomach, duodenum and testicles were collected for the comet assay. The liver, glandular stomach and duodenum for the comet assay was placed into ice-cold mincing and/or homogenization buffer (DPBS) and stored on ice. Tissues were rinsed sufficiently with cold mincing buffer to remove residual blood and stored in ice-cold mincing buffer until processed.

PREPARATION OF CELL NUCLEI OR SINGLE CELL
Single cell suspensions were prepared by chopping the tissue with scalpels in a small amount of ice-cold mincing solution. The scraped tissue solution was transferred into a homogenization tube and then gently homogenized using a homogenizer. Homogenate was filtered through a 70-micron pore nylon mesh and was then centrifuged at 800xg for 10 minutes. Single cells were suspended in DPBS. The collected testis was subjected to incision over the epithelial capsule of the testis to lead the seminiferous tubules squeezed out of the capsule. The germ cells were then collected by gently passing a tissue roller over the tubules at a suitable angle. Single cells were suspended in Dulbecco’s phosphate-buffered saline. The cell suspensions were maintained at 2 to 8 °C.

PREPARATION OF SLIDES
Three slides were prepared for each tissue from each animal and labelled with the study number, animal no., tissue, slide number (e.g., 1/3 to 3/3) and sex of each animal using pencil. To reduce the possibility of detachment of the agarose during the procedure, slides were pre-coated with 100 µL of liquid agarose and the agarose was allowed to dry to a thin film. Approximately 75 µL of cell suspension with 75 µL of 1.0 % low-melting agarose gel was mixed and rapidly pipetted onto the surface of the pre-coated slides and a coverslip was placed on it. Slides were placed on ice packs until the Agarose layer hardened (10 minutes). Slides were immersed in chilled lysing solution in the dark for overnight in a refrigerator under the light proof conditions. After completion of lysing, the slides were rinsed in distilled water to remove residual detergent and salts prior to alkali unwinding step.

UNWINDING, ELECTROPHORESIS AND STAINING
The slides were placed onto a platform of submarine-type electrophoresis unit containing a chilled electrophoresis solution. Buffer reservoirs were filled with freshly prepared Electrophoresis Buffer with pH 13.01 until the liquid level completely covers the slides. Slides were allowed to sit in the alkaline buffer for 20 minutes to allow for unwinding of the DNA and the expression of alkali-labile damage. Power supply ~0.9 V/cm was turned on and current was adjusted to 300 milliamperes by raising or lowering the buffer level. Electrophoresis solution was maintained at a constant temperature usually between 2 to 10 °C during electrophoresis under dimmed light. After completion of electrophoresis, power was turned off. Slides were removed from the buffer and place on a drain tray. The slides were immersed in the neutralization buffer for 10 minutes. All slides were dehydrated by immersing the slides into absolute ethanol up to 10 minutes. Slides were stained with 80 μL 1X ethidium bromide for 5 minutes and then dipped in chilled distilled water to remove excess stain. Drained slides were kept in cold 100 % methanol for 20 minutes for dehydration. Slides were air dried and then placed in an oven at 50±1 °C for 30 minutes.
Evaluation criteria:
All the slides were coded before evaluation to avoid group bias during evaluation. At least 150 cells were analysed per sample. The open comet software was used for analysis. The comet endpoints collected was % tail DNA, tail length in microns measured from the estimated edge of the head region closest to the anode. The frequency of hedgehogs was determined for at least 150 cells per sample.
Statistics:
Body weight and percentage of tail DNA were analysed by SPSS at a 95 % level of confidence (p <0.05) of significance*. Inter group comparison of body weight of day 1 day 2 and percent tail DNA comparison between treated and control groups. The statistical significances were designated by the superscripts as given below throughout the tables:
*: Statistically significant (p <0.05).
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid

TABLE 1 INDIVIDUAL ANIMAL CLINICAL SIGNS AND MORTALITY

Sex

Group & Dose (mg/kg)

Animal No.

Day

1

2

3

am

pm

am

pm

am

am

Male

 

G1 & 0

Rg5006

N

N

N

N

N

N

Rg5007

N

N

N

N

N

N

Rg5008

N

N

N

N

N

N

Rg5009

N

N

N

N

N

N

Rg5010

N

N

N

N

N

N

Rg5011

N

N

N

N

N

N

G2 & 500

Rg5012

N

N

N

N

N

N

Rg5013

N

N

N

N

N

N

Rg5014

N

N

N

N

N

N

Rg5015

N

N

N

N

N

N

Rg5016

N

N

N

N

N

N

Rg5017

N

N

N

N

N

N

G3 & 1000

Rg5018

N

N

N

N

N

N

Rg5019

N

N

N

N

N

N

Rg5020

N

N

N

N

N

N

Rg5021

N

N

N

N

N

N

Rg5022

N

N

N

N

N

N

Rg5023

N

N

N

N

N

N

G4 & 2000

Rg5024

N

N

N

N

N

N

Rg5025

N

N

N

N

N

N

Rg5026

N

N

N

N

N

N

Rg5027

N

N

N

N

N

N

Rg5028

N

N

N

N

N

N

Rg5029

N

N

N

N

N

N

G5 &250 (EMS)

Rg5030

N

N

N

N

N

N

Rg5031

N

N

N

N

N

N

Rg5032

N

N

N

N

N

N

Rg5033

N

N

N

N

N

N

Rg5034

N

N

N

N

N

N

Rg5035

N

N

N

N

N

N

N: Normal, EMS: Ethyl methanesulfonate

TABLE 2. INDIVIDUAL ANIMAL BODY WEIGHT

Sex

Group & Dose (mg/kg)

Animal No.

Day 1

Day 2

Day 3

Male

G1 & 0

Rg5006

209.60

212.97

218.60

Rg5007

232.39

238.11

242.90

Rg5008

257.86

260.02

267.21

Rg5009

248.14

250.06

259.22

Rg5010

255.49

258.97

268.02

Rg5011

271.01

278.58

284.90

Mean

245.75

249.79

256.81

±SD

21.77

22.41

23.16

G2 & 500

Rg5012

212.11

229.86

220.70

Rg5013

220.40

217.65

211.60

Rg5014

250.66

253.11

250.17

Rg5015

256.15

259.13

255.15

Rg5016

261.81

259.96

259.01

Rg5017

275.82

278.3

274.19

Mean

246.16

249.67

245.14

±SD

24.77

22.11

24.02

G3 & 1000

Rg5018

212.48

219.56

210.46

Rg5019

223.09

220.13

212.31

Rg5020

240.64

245.69

240.10

Rg5021

253.65

252.18

245.11

Rg5022

264.54

265.51

259.01

Rg5023

278.69

281.65

271.75

Mean

245.52

247.45

239.79

±SD

25.09

24.68

24.64

G4 & 2000

Rg5024

220.29

218.90

211.90

Rg5025

225.71

227.99

221.11

Rg5026

248.30

248.61

242.12

Rg5027

251.88

253.00

249.01

Rg5028

263.96

261.69

254.75

Rg5029

275.39

275.58

268.18

Mean

247.59

247.63

241.18

±SD

21.37

21.07

21.14

G5 & 250

(EMS)

Rg5030

219.85

220.50

218.59

Rg5031

231.20

235.65

234.61

Rg5032

243.35

245.01

245.49

Rg5033

256.51

256.39

253.21

Rg5034

255.38

254.93

250.18

Rg5035

297.29

295.86

290.18

Mean

250.60

251.39

248.71

±SD

26.88

25.53

23.91

SD: Standard deviation, EMS: Ethyl methanesulfonate.

TABLE 3 INDIVIDUAL ANIMAL GROSS PATHOLOGY 

Sex

Group & Dose

(mg/kg)

Animal No.

Gross Pathology Findings

(Internal/External)

Males

 

G1 & 0

Rg5006

NAD

Rg5007

NAD

Rg5008

NAD

Rg5009

NAD

Rg5010

NAD

Rg5011

NAD

G2 & 500

Rg5012

NAD

Rg5013

NAD

Rg5014

NAD

Rg5015

NAD

Rg5016

NAD

Rg5017

NAD

G3 & 1000

Rg5018

NAD

Rg5019

NAD

Rg5020

NAD

Rg5021

NAD

Rg5022

NAD

Rg5023

NAD

G4 & 2000

Rg5024

NAD

Rg5025

NAD

Rg5026

NAD

Rg5027

NAD

Rg5028

NAD

Rg5029

NAD

G5 & & 250 (EMS)

Rg5030

NAD

Rg5031

NAD

Rg5032

NAD

Rg5033

NAD

Rg5034

NAD

Rg5035

NAD

NAD: No Abnormalities Detected, EMS: Ethyl methanesulfonate.

TABLE 5 INDIVIDUAL ANIMAL % TAIL DNA

Sex

Group & Dose (mg/kg)

Animal No.

Organ

Liver

(% tail DNA)

Glandular Stomach

(% tail DNA)

Dodenum

(% tail DNA)

Males

G1 & 0

 

Rg5006

2.71

4.40

3.06

Rg5007

3.19

3.44

3.71

Rg5008

3.70

3.71

3.07

Rg5009

3.52

2.79

3.27

Rg5010

2.80

3.00

2.78

Rg5011

3.35

3.15

2.55

Mean

3.21

3.41

3.07

±SD

0.39

0.58

0.34

G2 & 500

 

Rg5012

3.66

3.24

4.17

Rg5013

2.86

3.57

2.58

Rg5014

4.14

3.02

3.72

Rg5015

3.97

3.82

3.97

Rg5016

3.95

5.84

5.21

Rg5017

3.66

4.57

5.30

Mean

3.70

4.01

4.16

±SD

0.45

1.05

1.01

G3 & 1000

 

Rg5018

2.96

5.04

3.27

Rg5019

3.46

3.42

4.09

Rg5020

3.95

2.46

4.29

Rg5021

3.77

4.10

5.05

Rg5022

5.36

4.99

5.43

Rg5023

4.42

3.82

3.44

Mean

3.99

3.97

4.26

±SD

0.83

0.98

0.86

G4 & 2000

 

 

Rg5024

4.08

2.96

2.43

Rg5025

3.72

3.76

4.83

Rg5026

3.48

5.48

3.90

Rg5027

3.58

4.77

5.49

Rg5028

3.79

4.98

3.30

Rg5029

3.92

3.76

3.66

Mean

3.76

4.28

3.94

±SD

0.22

0.94

0.99

G5 & 250

(EMS)

 

 

 

Rg5030

8.01

10.94

10.61

Rg5031

8.87

11.71

9.75

Rg5032

7.90

11.11

10.08

Rg5033

8.08

10.08

10.11

Rg5034

7.70

12.98

9.61

Rg5035

9.80

11.16

9.42

Mean

8.39*

11.33*

9.93*

±SD

0.80

0.94

0.43

*: Statistically significant at a 95 % level of confidence (p <0.05)

 

Conclusions:
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.
Executive summary:

The test item 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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Genotoxicity of the test item was assessed 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 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.


 


In vivo Micronucleus and Comet assay: 


The test item FAT 93504 was evaluated in the “In vivo Mammalian Alkaline Comet Assay” as per OECD Guideline No. 489, adopted on 29 July 2016 and "Mammalian Erythrocyte Micronucleus Test” as per OECD Guideline No. 474, adopted on 29 July 2016. 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 and 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.