CJ303

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

CJ303 was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 5 mg/plate in the absence and presence of S9 metabolic activation (OECD TG471).

CJ303 induced a significant level of chromosome aberrations in Chinese hamster V79 cells in the performed experiments with and without metabolic activation. The observed effects fulfilled all the criteria of positivity. Therefore, CJ303 was considered as clastogenic in this test system (OECD TG473).

CJ303 did not induce any Gene Mutation in the L5178Y cell up to 5000 µg/mL with or without metabolic activation. The result of CJ303 in vitro Mammalian Cell Gene Mutation test was negative (OECD TG476).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From October 4, 2018 to March 14, 2019

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)

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

CHO-K1

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

Based on the preliminary test result of cytotoxicity, the highest dose tested in this assay was 5000 ug/mL.

Vehicle / solvent:

Dimethylsulfoxide (DMSO)

Untreated negative controls:

yes

Remarks:

Ultrapure water

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

Based on the preliminary test result of cytotoxicity, the definite test were performed at 5000, 2000, 8000 and 320 µg/mL, and two replicates were prepared for each level. The groups were treated for 4 hours with metabolic activation, 4 hour without metabolic activation and 24 hours without metabolic activation.

Evaluation criteria:

1. Test Validity
When the PE0 of negative control = 60%-140%, PE2 of negative control = 40%-140%; MF of the positive control ≥ 2-fold the negative control value, this study will be considered valid.
2. Result Evaluation
A test item is consider positive if it meets both of the following criteria:
(1) At least one or more concentration produces a concentration-related increase in mutant frequency.
(2) MF of the test item ≥ 2-fold the negative control value.

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

CHO-K1

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

At the beginning and the end of treatment, no precipitate was observed in other dose.

Table 1. The Cytotoxicity Effect of CJ303 on L5178Y Cell

Treatment	Dose (µg/mL)	PE0 (%)	RS0 (%)	RSG (%)	RS2 (%)	RTG (%)
S9- 24h	Negative control	63.3	100.0	100.0	100.0	100.0
	Solvent control	60.7	95.9	132.5	96.9	128.4
	5000	50.3	79.5	43.3	91.5	39.6
	2000	58.0	91.6	102.4	105.1	107.6
	800	63.3	100.0	111.9	95.1	106.5
	320	65.8	104.0	118.4	91.3	108.2
	MMS	64.8	102.3	80.7	97.2	78.5
S9- 4h	Negative control	68.9	100.0	100.0	100.0	100.0
	Solvent control	62.2	90.4	75.6	104.2	78.8
	5000	63.2	91.7	94.1	89.6	84.3
	2000	69.7	101.2	159.8	76.5	122.2
	800	61.7	89.6	163.2	73.0	119.1
	320	67.7	98.3	122.9	94.5	116.1
	MMS	53.2	77.3	147.9	81.2	120.1
S9+ 4h	Negative control	62.0	100.0	100.0	100.0	100.0
	Solvent control	61.5	99.2	126.7	99.4	126.0
	5000	50.9	82.1	82.2	99.4	81.8
	2000	58.0	93.6	88.8	112.2	99.6
	800	63.1	101.7	96.5	113.9	109.9
	320	67.9	109.5	85.7	112.2	96.2
	CP	60.7	97.9	134.6	98.6	132.7

Table 2. The mutant frequencies of CJ303 on L5178Y Cell

Treatment	Dose (µg/mL)	PE2 (%)	MF (10^-6)	SC (%)
S9- 24h	Negative control	62.7	83.4	58.3
	Solvent control	60.7	120.0	-
	5000	57.4	86.0	-
	2000	65.9	94.9	-
	800	59.6	92.5	-
	320	57.2	119.3	-
	MMS	60.9	488.4	72.9
S9- 4h	Negative control	67.8	77.2	67.5
	Solvent control	70.6	93.0	-
	5000	60.7	74.3	-
	2000	51.8	92.6	-
	800	49.5	97.1	-
	320	64.0	116.0	-
	MMS	55.0	524.3	78.2
S9+ 4h	Negative control	60.5	80.3	69.3
	Solvent control	60.2	82.0	-
	5000	60.2	77.3	-
	2000	67.9	85.5	-
	800	68.9	94.8	-
	320	67.9	72.7	-
	CP	59.7	522.8	72.5

Table 3-1. The count result of PE0 and PE2 (S9- 24h)

Dose (µg/mL)	Cell number	PE0		PE2
		EW	TW	EW	TW
Negative control	011	33	96	36	96
	012	32	96	33	96
	021	40	96	35	96
	022	35	96	37	96
Solvent control	D1	33	96	33	96
	D2	40	96	40	96
5000	41	46	96	35	96
	42	40	96	42	96
2000	31	40	96	35	96
	32	36	96	32	96
800	21	32	96	38	96
	22	38	96	36	96
320	11	43	96	41	96
	12	33	96	36	96
MMS	M1	40	96	32	96
	M2	29	96	41	96

Table 3-2. The count result of PE0 and PE2 (S9- 4h)

Dose (µg/mL)	Cell number	PE0		PE2
		EW	TW	EW	TW
Negative control	011	30	96	31	96
	012	29	96	33	96
	021	34	96	35	96
	022	35	96	31	96
Solvent control	D1	34	96	31	96
	D2	37	96	31	96
5000	41	37	96	40	96
	42	33	96	33	96
2000	31	30	96	45	96
	32	33	96	39	96
800	21	40	96	44	96
	22	32	96	43	96
320	11	33	96	33	96
	12	32	96	36	96
MMS	M1	39	96	36	96
	M2	43	96	44	96

Table 3-3. The count result of PE0 and PE2 (S9+ 4h)

Dose (µg/mL)	Cell number	PE0		PE2
		EW	TW	EW	TW
Negative control	011	35	96	37	96
	012	37	96	34	96
	021	31	96	36	96
	022	40	96	39	96
Solvent control	D1	39	96	38	96
	D2	33	96	35	96
5000	41	43	96	42	96
	42	42	96	32	96
2000	31	40	96	35	96
	32	36	96	30	96
800	21	35	96	35	96
	22	35	96	29	96
320	11	35	96	35	96
	12	30	96	30	96
CP	C1	40	96	39	96
	C2	33	96	35	96

Conclusions:

It was concluded that when tested up to 5000 µg/mL with or without metabolic activation, CJ303 did not induce any Gene Mutation in the L5178Y cell. The result of CJ303 in vitro Mammalian Cell Gene Mutation test was negative.

Executive summary:

The mutagenic potential of CJ303 was assessed in the Mouse Lymphoma Cells (L5178Y), which can detect induced gene mutation.

 

L5178Y cells were exposed to CJ303 at the doses of 320µg/mL, 800µg/mL, 2000µg/mL and 5000µg/mL with metabolic activation for 4 hours, without metabolic activation for 4 hours and without metabolic activation for 24 hours, respectively. Dimethylsulfoxide (DMSO) was used as solvent control substances. Ultrapure water was used as negative control substances. Methyl methanesulfonate (MMS) and Cyclophosphamide (CP) were used as positive control substances for experiments with and without metabolic activation, respectively. After the treatment period, cytotoxicity was evaluated by the relative survival (relative to the negative control). All the culture were treated by trifluorothymidine (TF) to detect gene mutations.

 

When tested up to 5000µg/mL with or without metabolic activation, CJ303 did not induce any Gene Mutation in the L5178Y cell. The result of CJ303 in vitro Mammalian Cell Gene Mutation test was negative.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From July 11, 2016 to November 12, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Vehicle / solvent:

1g CJ303 was completely mixed with 4 mL DMSO and sterile water was added into solution up to 20 ml that the concentration contained 50 mg/mL.

Untreated negative controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

sodium azide

benzo(a)pyrene

mitomycin C

other:

Details on test system and experimental conditions:

Strains: The Salmonella typhimurium strains of TA98, TA100, TA102 TA1535and TA1537 were purchased from MOLTOX, Molecular Toxicology, Inc.
Culture medium: OXOID NB No.2
Incubation condition: 35±1℃

Evaluation criteria:

1. For the groups treated with S9 Mix, the range of natural reverse mutant colony number for TA98 was 32~51 CFU/plate, TA100 was 197~386 CFU/plate, TA102 was 378~487 CFU/plate, TA1535 was 18~41 CFU/plate and TA1537 was 9~26 CFU/plate.
2. For the groups not treated with S9 Mix, the range of natural reverse mutant colony number for TA98 was 22~51 CFU/plate, TA100 was 166~326 CFU/plate, TA102 was 262~407 CFU/plate, TA1535 was 17~43 CFU/plate and TA1537 was 6~25 CFU/plate.
3. Positive response criteria: At least one of the dosages should demonstrate a reverse mutation value double or higher times than that of the negative control group, and was calculated by SPSS, p<0.05 (One-way ANOVA), or test groups should present dose-response relationships. Those are judged the positive response.
4. The negative response criterion: In the bacterial reverse mutation test, the test results for the various dosage groups failed to comply with the positive response criteria are judged the negative response.

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Table 1. Characteristics of five Salmonella typhimurium strains

Test strain	Histidine requirement	uvrB mutation	rfa mutation	Ampicillin resistance
TA98	+	+	+	+
TA100	+	+	+	+
TA102	+	ϴ	+	+
TA1535	+	+	+	ϴ
TA1537	+	+	+	ϴ

+ means had the characteristic; ϴ means did not have the characteristic

Table 2. Toxicity of the test article of Salmonella typhimurium TA100

Group	Test article (mg/plate)	Reverse mutant colony number (CFU/plate)
With S9 Mix	Negative control group	212
	Positive control group^a	1,277
	Test group
	5	195
	2.5	178
	1.25	209
	0.625	202
	0.3125	253
Without S9 Mix	Negative control group	264
	Positive control group	554
	Test group
	5	208
	2.5	201
	1.25	217
	0.625	219
	0.3125	247

^a Positive control group: With S9 Mix: 2-Aminoanthracene (4.0μg/plate).

Without S9 Mix: Sodium azide (5μg/plate).

Table 3. Reverse mutation test of Salmonella typhimurium TA98

Group	Test article (mg/plate)	Reverse mutant colony number (CFU/plate)			Average of colonies^a
		1	2	3
With S9 Mix	Negative control group	37	45	48	43 ± 6
	Positive control group^b	398	408	415	407 ± 9^*
	Test group
	5	33	42	41	39 ± 5
	2.5	37	44	36	39 ± 4
	1.25	43	40	49	44 ± 5
	0.625	39	50	44	44 ± 6
	0.3125	55	50	44	50 ± 6
Without S9 Mix	Negative control group	29	26	28	28 ± 2
	Positive control group	296	282	236	271 ± 31^*
	Test group
	5	21	39	26	29 ± 9
	2.5	39	26	28	31 ± 7
	1.25	31	37	32	33 ± 3
	0.625	35	37	39	37 ± 2
	0.3125	47	48	25	40 ± 13

^a Average of colonies was shown as Mean ± S.D., the data were triplicate.

^b Positive control group: With S9 Mix: Benzo [a] pyrene (4.0μg/plate).

Without S9 Mix: 4-Nitroquinoline-N-oxide (0.5μg/plate).

^* Reverse mutant colony number were twice more than negative control group, ρ < 0.05 (One-Way ANOVA).

Table 4. Reverse mutation test of Salmonella typhimurium TA100

Group	Test article (mg/plate)	Reverse mutant colony number (CFU/plate)			Average of colonies^a
		1	2	3
With S9 Mix	Negative control group	292	288	221	267 ± 40
	Positive control group^b	1,295	1,358	1,280	1,311 ± 41^*
	Test group
	5	203	171	213	196 ± 22
	2.5	214	206	217	212 ± 6
	1.25	193	230	262	228 ± 35
	0.625	228	213	271	237 ± 30
	0.3125	304	290	284	293 ± 10
Without S9 Mix	Negative control group	219	272	265	252 ± 29
	Positive control group	521	592	639	584 ± 59^*
	Test group
	5	174	178	199	184 ± 13
	2.5	224	200	218	214 ± 12
	1.25	245	238	214	232 ± 16
	0.625	216	202	200	206 ± 9
	0.3125	249	266	270	262 ± 11

^a Average of colonies was shown as Mean ± S.D., the data were triplicate.

^b Positive control group: With S9 Mix: 2-Aminoanthracene (4.0μg/plate).

Without S9 Mix: Sodium azide (5μg/plate).

^* Reverse mutant colony number were twice more than negative control group, ρ < 0.05 (One-Way ANOVA).

Table 5. Reverse mutation test of Salmonella typhimurium TA102

Group	Test article (mg/plate)	Reverse mutant colony number (CFU/plate)			Average of colonies^a
		1	2	3
With S9 Mix	Negative control group	462	441	435	446 ± 14
	Positive control group^b	924	916	911	917 ± 7^*
	Test group
	5	438	451	424	438 ± 14
	2.5	440	399	458	432 ± 30
	1.25	449	437	430	439 ± 10
	0.625	448	425	455	443 ± 16
	0.3125	458	467	452	459 ± 8
Without S9 Mix	Negative control group	402	459	363	408 ± 48
	Positive control group	874	869	949	897 ± 45^*
	Test group
	5	346	390	370	369 ± 22
	2.5	440	386	418	415 ± 27
	1.25	440	391	403	411 ± 26
	0.625	403	399	412	405 ± 7
	0.3125	440	452	416	436 ± 18

^a Average of colonies was shown as Mean ± S.D., the data were triplicate.

^b Positive control group: With S9 Mix: 2-Aminoanthracene (10μg/plate).

Without S9 Mix: Mitomycin C (0.5μg/plate).

^* Reverse mutant colony number were twice more than negative control group, ρ < 0.05 (One-Way ANOVA).

Table 6. Reverse mutation test of Salmonella typhimurium TA1535

Group	Test article (mg/plate)	Reverse mutant colony number (CFU/plate)			Average of colonies^a
		1	2	3
With S9 Mix	Negative control group	19	19	31	23 ± 7
	Positive control group^b	192	259	263	238 ± 40^*
	Test group
	5	26	17	22	22 ± 5
	2.5	29	24	19	24 ± 5
	1.25	31	26	21	26 ± 5
	0.625	23	24	21	23 ± 2
	0.3125	26	22	21	23 ± 3
Without S9 Mix	Negative control group	29	26	27	27 ± 2
	Positive control group	299	240	276	372 ± 30^*
	Test group
	5	19	27	29	25 ± 5
	2.5	25	37	16	26 ± 11
	1.25	29	19	19	22 ± 6
	0.625	25	22	25	24 ± 2
	0.3125	30	40	40	37 ± 6

^a Average of colonies was shown as Mean ± S.D., the data were triplicate.

^b Positive control group: With S9 Mix: 2-Aminoanthracene (4.0μg/plate).

Without S9 Mix: Sodium azide (0.4μg/plate).

^* Reverse mutant colony number were twice more than negative control group, ρ < 0.05 (One-Way ANOVA).

Table 7. Reverse mutation test of Salmonella typhimurium TA1537

Group	Test article (mg/plate)	Reverse mutant colony number (CFU/plate)			Average of colonies^a
		1	2	3
With S9 Mix	Negative control group	20	15	23	19 ± 4
	Positive control group^b	416	425	432	424 ± 8^*
	Test group
	5	24	15	12	17 ± 6
	2.5	17	17	16	17 ± 1
	1.25	18	19	15	17 ± 2
	0.625	18	20	16	18 ± 2
	0.3125	21	22	16	20 ± 3
Without S9 Mix	Negative control group	17	15	14	15 ± 2
	Positive control group	1,082	1,163	976	1,074 ± 94^*
	Test group
	5	10	13	10	11 ± 2
	2.5	13	12	12	12 ± 1
	1.25	16	14	12	14 ± 2
	0.625	25	15	16	19 ± 6
	0.3125	12	14	14	13 ± 1

^a Average of colonies was shown as Mean ± S.D., the data were triplicate.

^b Positive control group: With S9 Mix: 2-Aminoanthracene (4.0μg/plate).

Without S9 Mix: 9-Aminoacridine (50.0μg/plate).

^* Reverse mutant colony number were twice more than negative control group, ρ < 0.05 (One-Way ANOVA).

Conclusions:

Activated with or without S9 Mix each concentration of CJ303, reverse mutant colony number of five testing strains which were TA98, TA100, TA102, TA1535 and TA1537, were not twice more than negative control groups. Therefore, test article CJ303 under the condition of bacterial reverse mutation test, the test result was negative.

Executive summary:

The bacterial reverse mutation test was conducted according to OECD 471:1997 to evaluate in vitro mutagenicity caused by CJ303. Test strains included Salmonella typhimurium TA98, TA100, TA102, TA1535 and TA1537. Negative control group, positive control group and test groups of each strain needed to activate with and without S9 Mix to test. In test for toxicity of the test article, detected toxicity of 5, 2.5, 1.25, 0.625 and 0.3125 mg/plate test article to TA100. The result showed that dose of 5 mg/plate did not cause toxicity. Therefore, according to OECD 471:1997, if 5 mg/plate test article did not cause toxicity, then choose 5 mg/plate as the highest concentration of the test, and used this concentration to dilute two-fold serial as 2.5, 1.25, 0.625 and 0.3125 mg/plate. The result of bacterial reverse mutation test showed that positive control group activated with and without S9 Mix could produce significant mutagenicity. No positive criteria were observed when the reverse mutant colonies of all test groups compared with the negative control group, and all test data were within the effective data range. These results indicated that CJ303, treated or not treated with S9m would not occur mutagenicity on this test condition.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From March 22, 2017 to Octobor 09, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

CHO-K1

Metabolic activation:

with and without

Metabolic activation system:

A cofactor-supplemented post-mitochondrial S9 fraction prepared from activated rat liver was used as an appropriate metabolic activation system.

Untreated negative controls:

yes

Remarks:

Distilled water

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

CHO-K1

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Table 1. Summary table of Chromosome Aberration Assay 1 without metabolic activation

Concentration (μg/mL) [Number of analyzed cells]	Time of Treatment / Sampling	RICC^#(%)	Precipitate^##	Mean % aberrant cells^###
CJ303 without metabolic activation (-S9)
Untreated control	3h / 20h	93	-	NE
Negative (vehicle) control [300]	3h / 20h	100	-	2.3
2000 μg/mL [300]	3h / 20h	61	-^a	8.9***
666.7 μg/mL [300]	3h / 20h	80	-^a	3.3
222.2 μg/mL [300]	3h / 20h	88	-^a	1.3
74.07 μg/mL	3h / 20h	100	-^a	NE
24.69 μg/mL	3h / 20h	93	-^a	NE
Positive control [300]	3h / 20h	67	-	15.3***

Negative (vehicle) control: (Distilled water)

Positive control (-S9): Ethyl methanesulfonate, 1 μL/mL

NE: not evaluated

RICC: Relative Increase in Cell Counts

^a: discoloured medium / minimally discoloured medium

^#: compared to the negative (vehicle) control

^##: in the final treatment medium at the end of the treatment

^###: excluding gaps

***: p<0.001 comparing numbers of aberrant cells excluding gaps with corresponding negative control

Table 2. Summary table of Chromosome Aberration Assay 1 with metabolic activation

Concentration (μg/mL) [Number of analyzed cells]	Time of Treatment / Sampling	RICC^#(%)	Precipitate^##	Mean % aberrant cells^###
CJ303 with metabolic activation (+S9)
Untreated control	3h / 20h	105	-	NE
Negative (vehicle) control [300]	3h / 20h	100	-	2.7
2000 μg/mL [300]	3h / 20h	88	-^a	9.0***
666.7 μg/mL [300]	3h / 20h	88	-^a	4.3
222.2 μg/mL [300]	3h / 20h	92	-^a	2.0
74.07 μg/mL	3h / 20h	101	-^a	NE
24.69 μg/mL	3h / 20h	101	-^a	NE
Positive control [70]	3h / 20h	66	-	71.4***

Negative (vehicle) control: (Distilled water)

Positive control (+S9): Cyclophosphamide, 6 μg/mL

NE: not evaluated

RICC: Relative Increase in Cell Counts

^#: compared to the negative (vehicle) control

^## : in the final treatment medium at the end of the treatment

^###: excluding gaps

^a: discoloured / minimally discoloured medium

***: p<0.001 comparing numbers of aberrant cells excluding gaps with corresponding negative control

Table 3. Summary table of Chromosome Aberration Assay 2 without metabolic activation

Concentration (μg/mL) [Number of analyzed cells]	Time of Treatment / Sampling	RICC^#(%)	Precipitate^##	Mean % aberrant cells^###
CJ303 without metabolic activation (-S9)
Untreated control	20h / 20h	94	-	NE
Negative (vehicle) control [300]	20h / 20h	100	-	4.3
222.2 μg/mL	20h / 20h	25	-^a	NE
74.07 μg/mL	20h / 20h	42	-^a	NE
24.69 μg/mL	20h / 20h	41	-^a	NE
8.23 μg/mL	20h / 20h	45	-^a	NE
2.74 μg/mL [185]	20h / 20h	46	-^a	27.03***
0.914 μg/mL [300]	20h / 20h	76	-	12.0***
0.305 μg/mL [300]	20h / 20h	78	-	7.3
0.102 μg/mL [300]	20h / 20h	95	-	6.0
0.0339 μg/Ml	20h / 20h	90	-	NE
Positive control [71]	20h / 20h	50	-	70.4***

Negative (vehicle) control: (Distilled water)

Positive control (-S9): Ethyl methanesulfonate, 0.4 μL/mL

NE: not evaluated

RICC: Relative Increase in Cell Counts

^#: compared to the negative (vehicle) control

^##: in the final treatment medium at the end of the treatment

^###: excluding gaps

^a: discoloured / minimally discoloured medium

***: p<0.001 comparing numbers of aberrant cells excluding gaps with corresponding negative control

Table 4. Summary table of Chromosome Aberration Assay 2 with metabolic activation

Concentration (μg/mL) [Number of analyzed cells]	Time of Treatment / Sampling	RICC^#(%)	Precipitate^##	Mean % aberrant cells^###
CJ303 with metabolic activation (+S9)
Untreated control	3h / 20h	92	-	NE
Negative (vehicle) control [300]	3h / 20h	100	-	3.7
2000 μg/mL [155]	3h / 20h	61	-^a	32.3***
666.7 μg/mL [290]	3h / 20h	86	-^a	15.9***
222.2 μg/mL [300]	3h / 20h	88	-^a	5.3
74.07 μg/mL	3h / 20h	91	-^a	NE
24.69 μg/mL	3h / 20h	95	-^a	NE
Positive control [80]	3h / 20h	69	-	62.5*

Negative (vehicle) control: (Distilled water)

Positive control (+S9): Cyclophosphamide, 6 μg/mL

NE: not evaluated

RICC: Relative Increase in Cell Counts

^#: compared to the negative (vehicle) control

^##: in the final treatment medium at the end of the treatment

^###: excluding gaps

^a: discoloured / minimally discoloured medium

***: p<0.001 comparing numbers of aberrant cells excluding gaps with corresponding negative control

Conclusions:

CJ303 induced a significant level of chromosome aberrations in Chinese hamster V79 cells in the performed experiments with and without metabolic activation. The observed effects fulfilled all the criteria of positivity. Therefore, CJ303 was considered as clastogenic in this test system.

Executive summary:

CJ303 was tested in vitro in a Chromosome Aberration Assay using Chinese hamster V79 lung cells. The test item was formulated in distilled water and it was examined up to cytotoxic concentrations according to OECD473. The treatment concentrations caused significant increases in the number of cells with structural chromosome aberrations in Assay 1 and Assay 2 with and without metabolic activation when compared with the appropriate negative (vehicle) control values. The increases were concentration-related and they were reproducible between the duplicate cultures. Based on these facts, the test item caused reproducible, concentration-related and statistically significant increases in the frequency of aberrant metaphases in V79 Chinese hamster cells, both in the absence and presence of S9-mix in this study. The negative (vehicle) control data were within the acceptable range for the spontaneous aberration frequency, the positive control substances caused a statistically significant increase in the number of structural aberrations excluding gaps in the experiments with or without metabolic activation demonstrating the sensitivity of the test system. The evaluated concentration range was considered to be adequate; at least three test item treated concentrations were evaluated in each assay. The tests were considered to be valid.

In conclusion, CJ303 induced a significant level of chromosome aberrations in Chinese hamster V79 cells in the performed experiments with and without metabolic activation. The observed effects fulfilled all the criteria of positivity. Therefore, CJ303 was considered as clastogenic in this test system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

CJ303 showed negative reaction for rodent peripheral blood micronucleus test under the conditions designed for this study, no mutagenic potential (OECD TG474).

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From October 7, 2019 to Decenber 13, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

ICR

Sex:

male

Details on test animals or test system and environmental conditions:

- Animal species: 25 of 6 weeks male ICR mice.
- Source: BioLASCO Taiwan Co., Ltd
- Temperature: 22±3℃
- Relative humidity: 55±15%
- Light cycle: 12 hours light and 12 hours dark
- Animal feeding situation: 5 mice per cage. Frequency of ventilation: 10~15 times/hour.
- Drinking water: Reverse osmosis water provided with water bottle.

Route of administration:

oral: gavage

Vehicle:

Test article was prepared with reverse osmosis water to 200.0000, 100.0000 and 50.0000 mg/mL, respectively.

Details on exposure:

The dosing volume of negative control and test article was 10 mL/kg BW by intraperitoneal injections.

Duration of treatment / exposure:

72 hours

Frequency of treatment:

During study period, the clinical observation of the mice was conducted every day. The body weights of the mice were determined before getting started with administration (Day 1) and 72 hours after the administration (Day 4).

Post exposure period:

48 and 72 hours after the test article administration, 5 µL of blood were collected from submandibular. The blood specimens were placed on slides that pre-dyed with acridine orange (1 mg/mL) and covered with coverslips. The blood were uniformly spread to a one layer blood cell thickness, and placed under room temperature for 3 hours protecting from light. Subsequently, fluorescence microscope was employed to observe the number of reticulocytes and micronucleus.

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

Positive control group

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Low dose group

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Medium dose group

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

High dose group

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Article name: Cyclophosphamide monohydrate (CPP)
Major ingredients: CPP, Form: Powder
Storage condition: Refrigeration (5±3℃)

Evaluation criteria:

1. Clinical observations were conducted during the study period and animal deaths should be documented.
2. The ratio of reticulocytes: Fluorescence microscope was employed to calculate the number of reticulocytes, stained with orange-red color, per 2,000 erythrocytes.
3. The micronucleus incidence: Fluorescence microscope was employed to calculate the number of micronucleated reticulocytes, micronuclei was stained with yellow-green color, per 4,000 reticulocytes.
4. The body weight of test and control groups were analyzed by using One-Way ANOVA of Duncan’s multiple range test in SPSS software. The ratio of reticulocyte and the micronucleus incidence of test and control groups were analyzed by using median and Mann-Whitney U test in SOSS software. A significant difference was defined as p< 0.05.
5. Positive criteria: The micronucleus incidence had statistic difference compared with negative control group, and showed dose-response relation.
1. Clinical observations were conducted during the study period and animal deaths should be documented.
2. The ratio of reticulocytes: Fluorescence microscope was employed to calculate the number of reticulocytes, stained with orange-red color, per 2,000 erythrocytes.
3. The micronucleus incidence: Fluorescence microscope was employed to calculate the number of micronucleated reticulocytes, micronuclei was stained with yellow-green color, per 4,000 reticulocytes.
4. The body weight of test and control groups were analyzed by using One-Way ANOVA of Duncan’s multiple range test in SPSS software. The ratio of reticulocyte and the micronucleus incidence of test and control groups were analyzed by using median and Mann-Whitney U test in SOSS software. A significant difference was defined as p< 0.05.
5. Positive criteria: The micronucleus incidence had statistic difference compared with negative control group, and showed dose-response relation.

Statistics:

Quality control: The ratio of reticulocyte and micronucleus in the negative control group at 48 hour and 72 hour should be in the range of laboratory historical data. The ratio of reticulocytes in the positive control group was significantly lower than negative control group and the micronucleus incidence was significantly higher than negative control group, then considered as effective data.

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

1. During study duration, results of body weight in all test groups showed no significantly different with negative control group (p>0.05) (Table 1). It indicated that test article had no influence on animal body weight.
2. The clinical observation results showed that testing mice of all groups were survival till the end of the study and no abnormal clinical sign were observed (Table 2).
3. The results of reticulocytes observation in 48 hours showed that the reticulocytes ratio median of negative control group was 56.0‰. The positive control group was 20.5‰ and significantly lower than negative control group (p<0.05). The low dose and medium dose group were 53.5‰ and 54.0‰ in reticulocytes ratio median, respectively. There were no significant difference compared with negative control group (p>0.05). The high dose group was 45.5‰ in reticulocytes ratio median and significantly lower than negative control group (p<0.05) (Table 3).
4. The results of micronucleus observation in 48 hours showed that the micronucleus incidence median of negative control group was 0.3‰. The positive control group was 10.5‰ and significantly higher than negative control group (p<0.05). The low dose, medium dose, and high dose group were 0.5‰, 0.5‰ and 0.5‰ in micronucleus incidence median respectively. There were no significant difference compared with negative control group (p>0.05) (Table 3).
5. The results of reticulocytes observation in 72 hours showed that the reticulocytes ratio median of negative control group was 56.0‰. The positive control group was 19.0‰ and significantly lower than negative control group (p<0.05). The low dose and medium dose group were 52.5‰ and 52.5‰ in reticulocytes ratio median, respectively. There were significantly lower than negative control group (p<0.05). The high dose group was 51.0‰ in reticulocytes ratio median. Tjere was no significant difference compared with negative control group (p<0.05) (Table 3).
6. The results of micronucleus observation in 72 hours showed that the micronucleus incidence median of negative control group was 0.5‰. The positive control group was 10.8‰ and significantly higher than negative control group (p<0.05). The low dose, medium dose, and high dose group were 0.3‰, 0.3‰ and 0.3‰ in micronucleus incidence median, respectively. There were no significant difference compared with negative control group (p>0.05) (Table 3).

Table 1. The body weight of testing mice

Group	Dose (mg/kg BW)	Body weight (g)^a
		Day 1	Day 4
Negative control group	-	28.2±0.8	29.6±0.8
Positive control group	50	28.5±0.8	30.0±0.9
Test group
Low dose group	500	28.6±1.4	30.1±1.0
Medium dose group	1000	29.3±1.0	30.9±1.7
High dose group	2000	28.5±1.2	30.0±1.4

^aAll data were shown as mean±SD, n=5.

Table 2. Clinical observation of testing mice

Group	Dose (mg/kg BW)	Daily Clinical Observation
		Day 1	Day 2	Day 3	Day 4
Negative control group	-	N	N	N	N
Positive control group	50	N	N	N	N
Test group
Low dose group	500	N	N	N	N
Medium dose group	1000	N	N	N	N
High dose group	2000	N	N	N	N

^aN: Normal.

Table 3. Reticulocytes ratio and micronucleus incidence in peripheral blood of all groups mice

Group	Dose (mg/kg BW)	Reticulocytes ratio RETs/2000 RBCs (‰)^a		Micronucleus incidence Mn-RETs/4000 RBCs (‰)^b
		Median^c (Q1~Q3)	p value^d	Median (Q1~Q3)	p valued
48 hours
Negative control group	-	56.0(51.8~58.8)	-	0.3 (0.2~0.7)	-
Positive control group	50	20.5(14.5~21.8)	0.008*	10.5(10.2~11.1)	0.008*
Test group
Low dose group	500	53.5(49.8~57.3)	0.413	0.5(0.0~0.9)	0.833
Medium dose group	1000	54.0(51.5~56.8)	0.548	0.5(0.2~08)	0.810
High dose group	2000	45.5(44.5~48.3)	0.008*	0.5(0.0~0.9)	0.833
72 hours
Negative control group	-	56.0(54.3~59.5)	-	0.5(0.3~0.8)	-
Positive control group	50	19.0(16.8~21.3)	0.008*	10.8(10.4~13.4)	0.008*
Test group
Low dose group	500	52.5(50.8~53.8)	0.016*	0.3(0.2~0.7)	0.468
Medium dose group	1000	52.5(50.8~53.5)	0.016*	0.3(0.2~0.7)	0.468
High dose group	2000	51.0(50.3~59.5)	0.381	0.3(0.2~0.7)	0.468

^aReticulocytes ratio: Reticulocytes (RETs)/2000 red blood cells (RBCs), n=5

^bMicronucleus incidence: Reticulocytes with micronucleus (Mn-RETs) / 4000 reticulocytes (RETs), n=5.

^c Q1: first quartile; Q3: third quartile.

^d Analysis by Mann-Whitney U test.

*Significantly different compared with negative control group, p<0.05

Conclusions:

The reticulocytes ratio of high dose group at 48 hours and low and medium dose groups at 72 hours were significantly lower than negative control group, but the average value were in the range of laboratory historical data. Therefore, it did not affect the determination of genotoxicity in this test. The micronucleus incidence of low, medium and high dose groups were no significantly different with negative control group. In conclusion, the test article CJ303 showed negative reaction for rodent peripheral blood micronucleus test under the conditions designed for this study, no mutagenic potential.

Executive summary:

This study was conducted according to the OECD474:2016 to evaluate the mutagenicity of CJ303 by rodent peripheral blood micronucleus test. Testing system was male ICR mice. The study included negative control group, positive control group and three test groups including 500, 1000 and 2000 mg/kg BW. Five testing animal were used per group. In this study, blood specimens of all group mice were collected and prepared for blood slide after 48 and 72 hours of test article or control article administration. Using fluorescence microscope observed the reticulocytes and number of micronucleus. Results showed that the reticulocytes ratio of high dose group at 48 hours and low and medium dose groups at 72 hours were significantly lower than negative control group, but the average value were in the range of laboratory historical data. Therefore, it did not affect the determination of genotoxicity in this test. The micronucleus incidence of low, medium and high dose groups were no statistical different compared with negative control group. The reticulocytes ratio of positive group was significantly lower than negative control group and the micronucleus incidence of positive control group was significantly higher than negative control group. In conclusion, CJ303 showed negative reaction for rodent peripheral blood micronucleus test under the conditions designed for this study, no mutagenic potential.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

in vitro gene mutation study in bacteria

Based on the preliminary assay results, 5 mg/plate was set as the highest dose in this study. In the mutagenicity assay, five doses of CJ303 at 0.3125, 0.625, 1.25, 2.5 and 5 mg/plate, concurrent negative and strain-specific positive controls were tested in tester strains TA98, TA100, TA102, TA1535 and TA1537 in triplicate with or without S9 Mix activation. No cytotoxicity was observed in all five tester strains up to 5 mg/plate in the absence and presence of metabolite activations. Results showed that CJ303 did not increase the number of revertants in all five tester strains TA98, TA100, TA102, TA1535 and TA1537 up to 5 mg/plate either in the absence or in the presence of metabolite activation.

Based on the data obtained from this study, it was concluded that under the test condition, CJ303 was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 5mg/plate in the absence and presence of S9 metabolic activation.

in vitro cytogenicity / chromosome aberration study in mammalian cells

CJ303 was tested in vitro in a Chromosome Aberration Assay using Chinese hamster V79 lung cells. The test item was formulated in distilled water and it was examined up to cytotoxic concentrations according to OECD473. The treatment concentrations caused significant increases in the number of cells with structural chromosome aberrations in Assay 1 and Assay 2 with and without metabolic activation when compared with the appropriate negative (vehicle) control values. The increases were concentration-related and they were reproducible between the duplicate cultures. Based on these facts, the test item caused reproducible, concentration-related and statistically significant increases in the frequency of aberrant metaphases in V79 Chinese hamster cells, both in the absence and presence of S9-mix in this study. The negative (vehicle) control data were within the acceptable range for the spontaneous aberration frequency, the positive control substances caused a statistically significant increase in the number of structural aberrations excluding gaps in the experiments with or without metabolic activation demonstrating the sensitivity of the test system. The evaluated concentration range was considered to be adequate; at least three test item treated concentrations were evaluated in each assay. The tests were considered to be valid.

in vitro gene mutation study in mammalian cells

The mutagenic potential of CJ303 was assessed in the Mouse Lymphoma Cells (L5178Y), which can detect induced gene mutation. L5178Y cells were exposed to CJ303 at the doses of 320µg/mL, 800µg/mL, 2000µg/mL and 5000µg/mL with metabolic activation for 4 hours, without metabolic activation for 4 hours and without metabolic activation for 24 hours, respectively. Dimethylsulfoxide (DMSO) was used as solvent control substances. Ultrapure water was used as negative control substances. Methyl methanesulfonate (MMS) and Cyclophosphamide (CP) were used as positive control substances for experiments with and without metabolic activation, respectively. After the treatment period, cytotoxicity was evaluated by the relative survival (relative to the negative control). All the culture were treated by trifluorothymidine (TF) to detect gene mutations. When tested up to 5000µg/mL with or without metabolic activation, CJ303 did not induce any Gene Mutation in the L5178Y cell. The result of CJ303 in vitro Mammalian Cell Gene Mutation test was negative.

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

The reticulocytes ratio of high dose group at 48 hours and low and medium dose groups at 72 hours were significantly lower than negative control group, but the average value were in the range of laboratory historical data. Therefore, it did not affect the determination of genotoxicity in this test. The micronucleus incidence of low, medium and high dose groups were no significantly different with negative control group.