CJ309

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

CJ309 was not mutagenic in the reverse mutation analysis of Salmonella typhimuriumup to 5000μg/plate in the absence and presence of S9 metabolic activation.(OECD TG471).

CJ309 did not induce chromosome aberration in CHO cells in absence or presence of S9 metabolic activation (OECD TG473).

Based on the read across result, the 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 bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From February 18, 2016 to August 29, 2016

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

Target gene:

Bacterial gene reverse mutation

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:

The post-mitochondrial fraction (S9) prepared from Aroclor 1254-induced Sprague-Dawley rats

Untreated negative controls:

yes

Remarks:

sterile deionized water

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

mitomycin C

other: Acridine mutagen ICR 191 2-Aminofluorene 2-Aminoanthracene

Evaluation criteria:

Acceptable ranges of background revertants for five tester strains are:
Tester Strain Revertants
TA98 10-60
TA100 50-240
TA102 180-480
TA1535 5-45
TA1537 2-25

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. Genotype Confirmation Tests of Salmonella typhimurium Tester Strains

Genotype character	Phenotypic observation	Tester Strains
		TA98	TA100	TA102	TA1535	TA1537
Histidine requirement	Growing on biotin plate	-	-	-	-	-
	Growing on histidine/biotin plate	+	+	+	+	+
rfa mutation	Inhibition zone of crystal violet	+	+	+	+	+
△uvrB mutation	Growing on non UV-irradiated plate	+	+	+	+	+
	Growing on UV-irradiated plate	-	-	+	-	-
R-factor	Ampicillin resistance	+	+	+	-	-
Genotype confirmed		Passed	Passed	Passed	Passed	Passed

+: the presence

-: the absence

Table 2. Mutagenicity Test of CJ309 in Salmonella typhimurium Strains without S9 Metabolic Activation

Treatment (μg/plate)		Number of Revertant Colonies in Salmonella typhimurium
		TA98			TA100			TA102			TA1535			TA1537
replicate		1	2	3	1	2	3	1	2	3	1	2	3	1	2	3
Negative control^a	I^e	18	23	25	82	79	60	380	407	433	16	13	23	9	25	8
	M^f	22 ± 4			74 ± 12			407 ± 27			17 ± 5			14 ± 10
50	I^e	35	25	17	68	81	71	402	314	345	20	10	16	9	16	14
	M^f	26 ± 9			73 ± 7			354 ± 45			15 ± 5			13 ± 4
150	I^e	25	14	25	70	71	67	335	343	336	14	24	12	15	16	22
	M^f	21 ± 6			69 ± 2			338 ± 4			17 ± 6			18 ± 4
500	I^e	26	23	22	86	66	66	364	315	317	24	25	23	13	13	16
	M^f	24 ± 2			73 ± 12			332 ± 28			24 ± 1			14 ± 2
1500	I^e	29	24	24	71	67	70	399	324	364	22	16	14	19	17	8
	M^f	26 ± 3			69 ± 2			362 ± 38			17 ± 4			15 ± 6
5000	I^e	17	24	16	58	65	67	384	282	326	14	15	11	19	14	16
	M^f	19 ± 4			63±5			331 ± 51			13 ± 2			16 ± 3
Positive control^b	I^e	224	217	196	575	587	591	1218	1018	1182	380	355	416	171	143	140
	M^f	212^c± 15			584^c± 8			1139^c± 107			384^d± 31			151^d± 17

a: Negative control was sterile deionized water.

b: Positive controls: 1μg/plate 2-nitrofluorene for TA98         0.5 μg/plate sodium azide for TA100

  62.5μg/plate mitomycin C for TA102     0.1 μg/plate sodium azide for TA1535

  0.5μg/plate acridine mutagen ICR 191 for TA1537 

c: Greater than 2-fold negative control spontaneous revertants

d: Greater than 3-fold negative control spontaneous revertants

e: I: Number of revertants/plate is shown for each individual plate

f: M: The value of mean ± S.D. from triplicate plates of each treatment was calculated

Table 3. Mutagenicity Test of CJ309 in Salmonella typhimurium Strains with S9 Metabolic Activation

Treatment (μg/plate)		Number of Revertant Colonies in Salmonella typhimurium
		TA98			TA100			TA102			TA1535			TA1537
replicate		1	2	3	1	2	3	1	2	3	1	2	3	1	2	3
Negative control^a	I^e	46	49	44	73	87	78	344	427	377	10	8	10	13	10	13
	M^f	46 ± 3			79 ± 7			383 ± 42			9 ± 1			12 ± 2
50	I^e	39	40	51	77	70	67	342	298	284	9	8	8	11	22	18
	M^f	43 ± 7			71 ± 5			308 ± 30			8 ± 1			17 ± 6
150	I^e	49	45	41	86	86	75	281	310	232	6	7	9	9	16	10
	M^f	45 ± 4			82 ± 6			274 ± 39			7 ± 2			12 ± 4
500	I^e	44	36	38	76	70	76	266	343	316	5	5	8	6	11	12
	M^f	39 ± 4			74 ± 3			308 ± 39			6 ± 2			10 ± 3
1500	I^e	48	37	35	80	65	64	324	454	364	8	12	10	13	8	19
	M^f	40 ± 7			70 ± 9			381 ± 67			10 ± 2			13 ± 6
5000	I^e	23	30	45	64	70	64	263	431	382	7	9	1	7	9	10
	M^f	33 ± 11			66 ± 3			359 ± 86			6 ± 4			9 ± 2
Positive control^b	I^e	248	229	225	610	651	642	1424	1350	1688	272	287	249	270	316	307
	M^f	234^c± 12			634^c± 22			1487^c± 178			269^d± 19			298^d± 24

a: Negative control was sterile deionized water.

b: Positive controls: 0.5μg/plate 2-aminofluorene for TA98     4 μg/plate 2-aminofluorene for TA100

  4μg/plate 2-aminoanthracene for TA102   1 μg/plate 2-aminoanthracene for TA1535

  2μg/plate 2-aminoanthracene for TA1537 

c: Greater than 2-fold negative control spontaneous revertants

d: Greater than 3-fold negative control spontaneous revertants

e: I: Number of revertants/plate is shown for each individual plate

f: M: The value of mean ± S.D. from triplicate plates of each treatment was calculated

Conclusions:

According to OECD 471 test method, CJ309 was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 5000 μg/plate.

Executive summary:

This test using the procedures outlined in the QPS Taiwan Study Plan for T65315029-GT which is based on the SOP for the OECD 471 (CTPS-TE00201) and OECD 471 (OECD,1997). The results of this OECD 471 test for CJ309 show that test validity criteria was met.

Based on the preliminary assay results, 5000μg/plate was set as the highest dose in this study. In the mutagenicity assay, five doses of CJ309 at 50, 150, 500, 1500 and 5000μg/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 5000μg/plate in the absence and presence of metabolite activations. Results showed that CJ309 did not increase the number of revertants in all five tester strains TA98, TA100, TA102, TA1535 and TA1537 up to 5000μg/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, CJ309 was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 5000μg/plate in the absence and presence of S9 metabolic activation.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From January 11, 2017 to June 07, 2017

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)

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:

The post-mitochondrial fraction (S9) of liver from Aroclor 1254 induced Sprague- Dawley rats

Untreated negative controls:

yes

Remarks:

Sterile deionized water

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

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. Karyology Analysis of Chinese Hamster Ovary Cells

No. of chromosome	<18	18	19	20	21	22	>22
No. of cells	0	0	9	35	5	1	0

Table 2. Concurrent Cytotoxicity Analysis of CJ309 in Chinese Hamster Ovary Cells

Concentration (μg/mL)	Cell Number (× 10^5cells)	ICC^a N-N0	RICC^b (%)	Cytotoxicity^c (%)
Before Treatment
Untreated (-S9)	30.4
Untreated (+S9)	26.4
After Treatment
Scheme I (-S9, 3h)
Negative Control	63.6	33.2	100.0	0.0
51.2	62.2	31.8	95.8	4.2
128	67.0	36.6	110.2	0.0
320	65.0	34.6	104.2	0.0
800	62.6	32.2	97.0	3.0
2000	61.2	30.8	92.8	7.2
Positive Control^d	48.6	18.2	54.8	45.2
Scheme II (+S9, 3h)
Negative Control	55.8	29.4	100.0	0.0
51.2	54.4	28.0	95.2	4.8
128	54.6	28.2	95.9	4.1
320	55.6	29.2	99.3	0.7
800	49.0	22.6	76.9	23.1
2000	52.8	26.4	89.8	10.2
Positive Control^e	44.6	18.2	61.9	38.1
Scheme III (-S9, 20h)
Negative Control	63.8	33.4	100.0	0.0
51.2	57.6	27.2	81.4	18.6
128	110.2	79.8	238.9	0.0
320	108.0	77.6	232.3	0.0
800	54.4	24.0	71.9	28.1
2000	58.8	28.4	85.0	15.0
Positive Control^f	56.8	26.4	79.0	21.0

a ICC: increased in cell counts = Cell No._{After treatment} (N) - Cell No._{Before treatment} (N₀)

b RICC: relative increase in cell counts; RICC = (ICC_treatment/ICC_control) × 100

c Cytotoxicity (%) = 100 – RICC

d Positive control was 0.33 μg/mL mitomycin C (MMC)

e Positive control was 11.2 μg/mL cyclophosphamide (CPP)

f Positive control was 0.2 μg/mL mitomycin C (MMC)

 

Table 3. Summary of Chromosome Aberrations in Chinese Hamster Ovary Cells for CJ309

Treatment	Concentration (μg/mL)	Treating Hour	S9 (-/+)	Aberrant Cells (%)
Scheme I (-S9, 3h)
Negative Control^a	0	3	-	0.33
Test Article	51.2	3	-	0.33
	128	3	-	0.67
	320	3	-	0.33
	800	3	-	0
	2000	3	-	1
Positive Control^c	0.33	3	-	19.33*
Scheme II (+S9, 3h)
Negative Control^b	0	3	+	0
Test Article	51.2	3	+	0.67
	128	3	+	0.67
	320	3	+	0.67
	800	3	+	1
	2000	3	+	0.33
Positive Control^d	11.2	3	+	24*
Scheme III (-S9, 20h)
Negative Control^a	0	20	-	0.33
Test Article	51.2	20	-	0.33
	128	20	-	0.67
	320	20	-	1
	800	20	-	1.33
	2000	20	-	−
Positive Control^e	0.2	20	-	29*

All data were scored from 300 metaphase cells of each treatment (duplicate cultures).

a: Negative control was 10% sterile deionized water in McCoy’s 5A medium.

b: Negative control was 10% sterile deionized water in S-9 mixture medium.

c: Positive control was 0.33 μg/mL mitomycin C (MMC).

d: Positive control was 11.2 μg/mL cyclophosphamide (CPP).

e: Positive control was 0.2 μg/mL mitomycin C (MMC).

*: The frequency of aberrant cells was significantly higher than that of the negative control (One-tailed binomial test, α = 0.01).

−: Too few metaphases

Conclusions:

According to OECD 473 test method, CJ309 did not induce chromosome aberration in CHO cells in absence or presence of S9 metabolic activation.

Executive summary:

This test using the procedures outlined in the QPS Taiwan Study Plan for T65316027-GT and OECD 473 (OECD, 2016). The results of this OECD 473 test for CJ309 show that test validity criteria was met.

A preliminary concentration-range finding cytotoxicity test was performed in three test schemes: 3-hour treatment in the absence and presence of S9 metabolic activation (Schemes I and II) and 20-hour continuous treatment in the absence of S9 metabolic activation (Scheme III), and the results suggested that a concentration of 2000 μg/mL in all three schemes would be used in the chromosome aberration assay. Based on the cytotoxicity result, five concentrations of 51.2, 128, 320, 800 and 2000 μg/mL were used for all three schemes in the chromosome aberration test. In test group, the cell proportions with abnormal chromosome in three Schemes were not higher than 3%. Based on the data obtained from this study, it was concluded that under the test condition,CJ309 did not induce chromosome aberration in CHO cells in absence or presence of S9 metabolic activation.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From September 11, 2018 to December 25, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to OECD guideline and in accordance with GLP

Justification for type of information:

Demonstrates that the hypothesis is supported by referring to a data set: all claims must be supported by data.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes

Type of assay:

other: in vitro mammalian cell gene mutation tests using Chinese Hamster ovary cell (CHO-K1)

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

Cell line and incubation condition:
1. Cell line: Chinese hamster ovary cells (CHO-K1, BCRC 60006) is purchased from the Food Industry Research and Development Institute, Taiwan.
2. Culture medium Ham’s F-12 medium (including 2.0 mM L-Glutamine) with 10% fetal bovine serum (FBS).
3. Incubation condition: 37±1℃ incubator with 5±1% CO2.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

1. Cell viability test:
The cell viability for each test group after treating with or without S9 Mix was analyzed (Table 1). When the dosage of test article was 2.0 mg/mL with or without S9 Mix for 3 hours, the cell viability was 60.85% and 74.63%. Therefore, 2.0 mg/L was used as the highest dosage for the gene mutation test.
2. Gene mutation test:
The colonies formation frequency and mutation frequency for each test group after treating with or without S9 Mix were analyzed (Table 2 &3). The results were as below:
2.1 After treating with S9 Mix for 3 hours, the negative control group mutation frequency was 19.0 x 10^-6, the positive control group (Benzo [a] pyrene) mutation frequency was 51.8 x 10^-6, that was significantly different from the negative control group (p<0.05). At test article concentrations of 2.0 mg/mL, 1.0 mg/mL, 0.5 mg/mL and 0.25 mg/mL, the mutation frequency were 37.3 x 10^-6, 36.7 x 10^-6, 39.9 x 10^-6 and 25.5 x 10^-6, was no significantly different from the negative control group (p> 0.05).
2.2 After treating without S9 Mix for 3 hours, the negative control group mutation frequency was 17.1 x 10^-6, the positive control group (4- Nitroquinoline-1-oxide) mutation frequency was 44.8 x 10^-6, that was significantly different from the negative control group (p<0.05). At test article concentrations of 2.0 mg/mL, 1.0mg/mL, 0.5mg/mL and 0.25 mg/mL, the mutation frequency were 21.1 x 10^-6, 21.4 x 10^-6, 20.9 x 10^-6 and 18.0 x 10^-6, was no significantly different from the negative control group (P>0.05) (Table3).

Remarks on result:

no mutagenic potential (based on QSAR/QSPR prediction)

Table 1. Cell viability analysis

Group	Test article	Average colony numbers ^a	Relative survival (%)^b
With S9 Mix	Negative control ^c	74.5±12.0	100.00±0.00
	Positive control ^d	65.0±5.7	85.98±0.07
	Test groups (mg/mL)
	2.0	46.0±9.9	60.85±0.13
	1.0	46.0±2.8	60.85±0.04
	0.5	50.5±9.2	66.80±0.12
	0.25	57.5±7.8	76.06±0.10
Without S9 Mix	Negative control	60.0±7.1	100.00±0.00
	Positive control	42.5±13.4	63.43±0.20
	Test groups (mg/mL)
	2.0	50.0±8.5	74.63±0.13
	1.0	45.5±4.9	67.91±0.07
	0.5	53.5±6.4	79.85±0.09
	0.25	56.0±4.2	83.58±0.06

^a Values were expressed as Mean±SD, and tests were repeated two times.

^b Relative survival = each colony numbers of the positive control or test groups / the average of colony numbers in the negative control x 100%, then calcultated the Mean±SD.

^c Negative control: Ham's F-12 medium with 10% FBS (S9 Mix or not)

^d Positive control: 4 µg/mL B[a]P for the cells treated with S9 Mix, and 0.25 µg/mL 4-NQD for the cells treated without S9 Mix.

Table 2. Colonies formation frequency analysis

Group	Test article	Average colony numbers ^a	Colonies formation frequency ^b
With S9 Mix	Negative control ^c	141.3±9.0	0.71
	Positive control ^d	115.7±13.7	0.58
	Test groups (mg/mL)
	2.0	157.7±25.5	0.79
	1.0	128.0±39.4	0.64
	0.5	103.7±62.9	0.52
	0.25	101.7±37.5	0.51
Without S9 Mix	Negative control	176.3±20.2	0.88
	Positive control	104.3±22.9	0.52
	Test groups (mg/mL)
	2.0	138.3±41.2	0.69
	1.0	122.7±17.4	0.61
	0.5	117.0±34.7	0.59
	0.25	115.3±46.9	0.58

^a Values were expressed as Mean±SD, and tests were repeated three times.

^b Colonies formation frequency = numbers of colonies / the number of seeding.

^c Negative control: Ham's F-12 medium with 10% FBS (S9 Mix or not)

^d Positive control: 4 µg/mL B[a]P for the cells treated with S9 Mix, and 0.25 µg/mL 4-NQD for the cells treated without S9 Mix.

Table 3. Mutation frequency analysis

Group	Test article	Average colony numbers ^a	Mutationfrequency (x 10^-6) ^b
With S9 Mix	Negative control ^c	13.3±7.0	19.0
	Positive control ^d	30.0±4.4	51.8
	Test groups (mg/mL)
	2.0	28.7±4.5	37.3
	1.0	22.7±6.5	36.7
	0.5	18.3±7.4	39.9
	0.25	12.3±4.7	25.5
Without S9 Mix	Negative control	15.3±7.4	17.1
	Positive control	25.0±15.1	44.8
	Test groups (mg/mL)
	2.0	13.7±3.5	21.1
	1.0	13.0±2.6	21.4
	0.5	11.0±2.0	20.9
	0.25	9.7±2.5	18.0

^a Values were expressed as Mean±SD, and tests were repeated three times.

^b Mutation frequency = (numbers of colonies / number of seeding) x (1/colonies formation frequency).

^c Negative control: Ham's F-12 medium with 10% FBS (S9 Mix or not)

^d Positive control: 4 µg/mL B[a]P for the cells treated with S9 Mix, and 0.25 µg/mL 4-NQD for the cells treated without S9 Mix.

^e Significantly different from the negative control group (p<0.05)

Conclusions:

This in vitro mammalian cell gene mutation test was performed in accordance with OECD 476:2015 guideline. The results indicated that the positive control group (Benzo[a]pyrene and 4-Nitroquinoline-1-oxide) mutation frequency was significantly different from the negative control group (p<0.05), meaning that this experiment was effective. The results by statistical analysis indicated that the mutation frequency of the test article for 3 hours treatment with S9 Mix and without S9 Mix was no significantly different from the negative control group. According to the above results, the in vitro mammalian cell gene mutation test was negative.

Executive summary:

This in vitro mammalian cell gene mutation test was conducted according to OECD 476:2015 guideline to evaluate the mutagenicity of CJ302. Chinese Hamster ovary cell (CHO-K1) was used in the experiment. The cytotoxicity of the test article for CHO-K1 was used to set the highest dosage and number of seeding cells in the gene mutation test. The gene mutation test processing conditions were treating with S9 Mix and without S9 Mix for 3 hours. The test article highest concentration was 2.0 mg/mL as determined by a cell viability test. By statistical analysis, the mutation frequency of each test group had no significant difference from the negative control group after the test article for 3 hours treatment with S9 Mix and without S9 Mix. Therefore, under this test conditions, the in vitro mammalian cell gene mutation test was negative.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Bacterial reverse mutation test (OECD TG471)

Based on the preliminary assay results, 5000μg/plate was set as the highest dose in this study. In the mutagenicity assay, five doses of CJ309 at 50, 150, 500, 1500 and 5000μg/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. The results of concurrent positive and negative controls and three non-cytotoxic dose levels obtained supported the validity of the assay.

No cytotoxicity was observed in all five tester strains up to 5000 μg/plate in the absence and presence of metabolite activations. Results showed that CJ309 did not increase the number of revertants in all five tester strains TA98, TA100, TA102, TA1535 and TA1537 up to 5000 μg/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, CJ309 was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 5000μg/plate in the absence and presence of S9 metabolic activation.

 

Mammalian chromosomal aberration test (OECD TG473)

A preliminary concentration-range finding cytotoxicity test was performed in three test schemes: 3-hour treatment in the absence and presence of S9 metabolic activation (Schemes I and II) and 20-hour continuous treatment in the absence of S9 metabolic activation (Scheme III), and the results suggested that a concentration of 2000 μg/mL in all three schemes would be used in the chromosome aberration assay. Based on the cytotoxicity result, five concentrations of 51.2, 128, 320, 800 and 2000 μg/mL were used for all three schemes in the chromosome aberration test. In test group, the cell proportions with abnormal chromosome in three Schemes were not higher than 3%. Based on the data obtained from this study, it was concluded that under the test condition, CJ309 did not induce chromosome aberration in CHO cells in absence or presence of S9 metabolic activation.

In vitro mammalian cell gene mutation test (OECD 476)

This in vitro mammalian cell gene mutation test was performed in accordance with OECD 476:2015 guideline. The results indicated that the positive control group (Benzo[a]pyrene and 4-Nitroquinoline-1-oxide) mutation frequency was significantly different from the negative control group (p<0.05), meaning that this experiment was effective. The results by statistical analysis indicated that the mutation frequency of the test article for 3 hours treatment with S9 Mix and without S9 Mix was no significantly different from the negative control group. According to the above results, the in vitro mammalian cell gene mutation test was negative.