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

Genetic toxicity: in vivo

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

254-959-7

EC Name:

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

Cas Number:

40530-60-7

Molecular formula:

C19H17NO7

IUPAC Name:

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

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

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

Test animals

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.

Administration / exposure

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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle control

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw/day

Remarks:

Low dose group

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Mid dose group

Doses / concentrations 4

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

Examinations

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 was 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. Inter group 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).

Results and discussion

Test results

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.

Any other information on results incl. tables

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

 

Applicant's summary and conclusion

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.