5-({4-chloro-6-[ethyl(phenyl)amino]-1,3,5-triazin-2-yl}amino)-4-hydroxy-3-[(1-sulfo-2-naphthyl)diazenyl]naphthalene-2,7-disulfonic acid, lithium sodium salts

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

CJ306 was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 5000μg/plate in the absence and presence of S9 metabolic activation(OECD TG471).

CJ306 induced a significant level of chromosome aberrations in Chinese hamster V79 cells in the 20-hour treatment without metabolic activation(OECD TG473).

CJ306 was negative effect under the condition of in vitro mammalian cell gene mutation test (OECD TG490).

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 April 20, 2016 to October 14, 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

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 typhimuriumTester Strains

Genotype character	Phenotypic observation	Tester Strains
		TA98	TA100	TA102	TA1535	TA1537
Histidine requirement	Growing on biotin plate	-	-	-	-	-
	Growing on histidine/biotin plate	+	+	+	+	+
rfamutation	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 CJ306 in Salmonella typhimurium Strains without S9 Metabolic Activation

Treatment (μg/plate)		Number of Revertant Colonies inSalmonella 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	13	41	17	62	80	65	326	312	374	19	10	13	8	9	6
	M^f	24 ± 15			69 ± 10			337 ± 33			14 ± 5			8 ± 2
50	I^e	25	41	38	66	67	54	317	247	274	13	11	12	12	6	4
	M^f	35 ± 9			62 ± 7			279 ± 35			12 ± 1			7 ± 4
150	I^e	29	32	20	83	80	64	218	274	400	11	12	19	10	4	6
	M^f	27 ± 6			76 ± 10			297 ± 93			14 ± 4			7 ± 3
500	I^e	19	27	22	62	67	68	257	309	302	20	17	15	4	11	12
	M^f	23 ± 4			66 ± 3			289 ± 28			17 ± 3			9 ± 4
1500	I^e	22	30	25	69	50	72	286	247	191	9	8	8	10	10	8
	M^f	26 ± 4			64 ± 12			241 ± 48			8 ± 1			9 ± 1
5000	I^e	21	27	24	66	50	68	249	272	251	9	15	13	9	6	5
	M^f	24 ± 3			61 ± 10			257 ± 13			12 ± 3			7 ± 2
Positive control^b	I^e	190	220	205	584	483	589	1061	1259	1060	370	425	410	78	90	110
	M^f	205^c± 15			552^c± 60			1127^c± 115			402^d± 28			93^d± 16

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 CJ306 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	21	18	29	89	78	90	453	461	416	11	13	9	10	5	10
	M^f	23 ± 6			86 ± 7			443 ± 24			11 ± 2			8 ± 3
50	I^e	23	16	12	103	82	87	385	418	385	9	6	9	6	7	12
	M^f	17 ± 6			91 ± 11			396 ± 19			8 ± 2			8 ± 3
150	I^e	26	20	21	98	81	80	409	396	431	9	8	7	9	9	14
	M^f	22 ± 3			86 ± 10			412 ± 18			8 ± 1			11 ± 3
500	I^e	23	22	22	85	91	89	391	392	408	14	14	12	16	10	4
	M^f	22 ± 1			88 ± 3			397 ± 10			13 ± 1			10 ± 6
1500	I^e	15	26	17	73	71	88	333	365	361	13	7	7	4	9	10
	M^f	19 ± 6			77 ± 9			353 ± 17			9 ± 3			8 ± 3
5000	I^e	17	24	23	74	71	84	329	308	306	13	13	9	6	11	7
	M^f	21 ± 4			76 ± 7			314 ± 13			12 ± 2			8 ± 3
Positive control^b	I^e	174	162	141	597	573	609	1968	2178	2076	152	167	180	205	183	209
	M^f	159^c± 17			593^c± 18			2074^c± 105			166^d± 14			199^d±14

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, CJ306 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 T65315028-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 CJ306 show that test validity criteria was met.

Based on the preliminary assay results, 5000μg/platewas set as the highest dose in this study. In the mutagenicity assay, five doses of CJ306 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 CJ306 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, CJ306 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 April 20, 2017 to November 20, 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 lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Untreated negative controls:

yes

Remarks:

Distilled water

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Table 1. Cytotoxicity Results of Chromosome Aberration Assay 1 experiment without metabolic activation

Test group	Dose (µg/mL)	S9-mix	Treatment / sampling time	Cell number (total)	RICC (%)*	Observations beginning / end of treatment
Untreated control	−	−	3/20	5.63E+06	103	normal / normal (pH: 7.2; osm: 344 mmol/kg)
Negative (vehicle) control	−	−	3/20	5.50E+06	100	normal / normal (pH: 7.2; osm: 342 mmol/kg)
CJ306	1250	−	3/20	1.50E+06	20	discoloured medium / discoloured medium (pH:**; osm: 356 mmol/kg)
	1000	−	3/20	2.55E+06	41	discoloured medium / discoloured medium (pH: **; osm: 362 mmol/kg)
	500	−	3/20	5.05E+06	91	discoloured medium / discoloured medium (pH: **; osm: 366 mmol/kg)
	250	−	3/20	5.38E+06	98	discoloured medium / discoloured medium (pH: **; osm: 352 mmol/kg)
	125	−	3/20	5.48E+06	100	discoloured medium / discoloured medium (pH: **; osm: 350 mmol/kg)
Positive control (1 µL/mL EMS)	−	−	3/20	3.88E+06	68	normal / normal (pH: 7.2; osm: 366 mmol/kg)

* compared to the negative (vehicle) control (Distilled water)

** The pH of these samples could not be measured with pH indicator paper due to the intensive blue colour of the samples.

RICC: Relative Increase in Cell Counts

osm: osmolality

Note: Duplicate counts were performed at each counting.

 

Table 2. Cytotoxicity Results of Chromosome Aberration Assay 1 experiment with metabolic activation

Test group	Dose (µg/mL)	S9-mix	Treatment / sampling time	Cell number (total)	RICC (%)*	Observations beginning / end of treatment
Untreated control	−	+	3/20	5.85E+06	102	normal / normal (pH: 7.2; osm: 351 mmol/kg)
Negative (vehicle) control	−	+	3/20	5.75E+06	100	normal / normal (pH: 7.2; osm: 349 mmol/kg)
CJ306	700	+	3/20	2.58E+06	40	discoloured medium / discoloured medium (pH:**; osm: 352 mmol/kg)
	500	+	3/20	2.50E+06	57	discoloured medium / discoloured medium (pH: **; osm: 341 mmol/kg)
	250	+	3/20	4.73E+06	80	discoloured medium / discoloured medium (pH: **; osm: 343 mmol/kg)
	125	+	3/20	4.65E+06	79	discoloured medium / discoloured medium (pH: **; osm: 343mmol/kg)
	62.5	+	3/20	5.18E+06	89	discoloured medium / discoloured medium (pH: **; osm: 346mmol/kg)
Positive control (6 µg/mL CP)	−	+	3/20	3.40E+06	55	normal / normal (pH: 7.2; osm: 355 mmol/kg)

* compared to the negative (vehicle) control (Distilled water)

** The pH of these samples could not be measured with pH indicator paper due to the intensive blue colour of the samples.

RICC: Relative Increase in Cell Counts

osm: osmolality

Note: Duplicate counts were performed at each counting.

 

Table 3. Cytotoxicity Results of Chromosome Aberration Assay 2 experiment without metabolic activation

Test group	Dose (µg/mL)	S9-mix	Treatment / sampling time	Cell number (total)	RICC (%)*	Observations beginning / end of treatment
Untreated control	−	−	20/20	7.53E+06	92	normal / normal (pH: 7.4; osm: 346 mmol/kg)
Negative (vehicle) control	−	−	20/20	8.13E+06	100	normal / normal (pH: 7.4; osm: 339 mmol/kg)
CJ306	700	−	20/20	1.15E+06	9	discoloured medium / discoloured medium (pH:**; osm: 339 mmol/kg)
	500	−	20/20	3.45E+06	39	discoloured medium / discoloured medium (pH: **; osm: 343 mmol/kg)
	250	−	20/20	5.13E+06	61	discoloured medium / discoloured medium (pH: **; osm: 338 mmol/kg)
	125	−	20/20	7.08E+06	86	discoloured medium / discoloured medium (pH: **; osm: 340 mmol/kg)
	62.5	−	20/20	7.18E+06	88	discoloured medium / discoloured medium (pH: **; osm: 336 mmol/kg)
Positive control (0.4 µL/mL EMS)	−	−	20/20	5.30E+06	63	normal / normal (pH: 7.4; osm: 340mmol/kg)

* compared to the negative (vehicle) control (Distilled water)

** The pH of these samples could not be measured with pH indicator paper due to the intensive blue colour of the samples.

RICC: Relative Increase in Cell Counts

osm: osmolality

Note: Duplicate counts were performed at each counting.

 

Table 4. Cytotoxicity Results of Chromosome Aberration Assay 2 experiment with metabolic activation

Test group	Dose (µg/mL)	S9-mix	Treatment / sampling time	Cell number (total)	RICC (%)*	Observations beginning / end of treatment
Untreated control	−	+	3/20	7.18E+06	107	normal / normal (pH: 7.4; osm: 337 mmol/kg)
Negative (vehicle) control	−	+	3/20	6.75E+06	100	normal / normal (pH: 7.4; osm: 331 mmol/kg)
CJ306	700	+	3/20	3.23E+06	44	discoloured medium / discoloured medium (pH:**; osm: 330 mmol/kg)
	500	+	3/20	5.55E+06	81	discoloured medium / discoloured medium (pH: **; osm: 334 mmol/kg)
	250	+	3/20	6.63E+06	98	discoloured medium / discoloured medium (pH: **; osm: 329 mmol/kg)
	125	+	3/20	6.65E+06	98	discoloured medium / discoloured medium (pH: **; osm: 329 mmol/kg)
	62.5	+	3/20	7.35E+06	110	discoloured medium / discoloured medium (pH: **; osm: 329 mmol/kg)
Positive control (6 µg/mL CP)	−	+	3/20	4.50E+06	64	normal / normal (pH: 7.4; osm: 334 mmol/kg)

* compared to the negative (vehicle) control (Distilled water)

** The pH of these samples could not be measured with pH indicator paper due to the intensive blue colour of the samples.

RICC: Relative Increase in Cell Counts

osm: osmolality

Note: Duplicate counts were performed at each counting.

 

Table 5. 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^###
CJ306 without metabolic activation (-S9)
Untreated control	3h / 20h	103	−	NE
Negative (vehicle) control [300]	3h / 20h	100	−	3.0
1250 µg/mL	3h / 20h	20	−^a	NE
1000 µg/mL [300]	3h / 20h	41	−^a	2.0
500 µg/mL [300]	3h / 20h	91	−^a	3.7
250 µg/Ml [300]	3h / 20h	98	−^a	1.7
125 µg/mL	3h / 20h	100	−^a	NE
Positive control [300]	3h / 20h	68	−	8.3**

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

#: 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 6. 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^###
CJ306 without metabolic activation (+S9)
Untreated control	3h / 20h	102	−	NE
Negative (vehicle) control [300]	3h / 20h	100	−	4.7
700 µg/mL [300]	3h / 20h	40	−^a	4.3
500 µg/mL [300]	3h / 20h	57	−^a	5.3
250 µg/mL [300]	3h / 20h	80	−^a	4.3
125 µg/mL	3h / 20h	79	−^a	NE
62.5 µg/mL	3h / 20h	89	−^a	NE
Positive control [82]	3h / 20h	55	−	61.0***

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 medium

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

 

Table 7. 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^###
CJ306 without metabolic activation (-S9)
Untreated control	20h / 20h	92	−	NE
Negative (vehicle) control	20h / 20h	100	−	1.7
700 µg/mL	20h / 20h	9	−^a	NE
500 µg/mL [300]	20h / 20h	39	−^a	11.0***
250 µg/mL [300]	20h / 20h	61	−^a	7.7***
125 µg/mL [300]	20h / 20h	86	−^a	7.0**
62.5 µg/mL	20h / 20h	88	−^a	NE
Positive control [127]	20h / 20h	63	−	39.4***

Negative (vehicle) control: Distilled water

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

NE: not evaluated

RICC: Relative Increase in Cell Counts

a: discoloured medium

#: compared to the negative (vehicle) control

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

###: excluding gaps

**: p<0.01 comparing numbers of aberrant cells excluding gaps with corresponding negative control

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

 

Table 8. 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 without metabolic activation (+S9)
Untreated control	3h / 20h	107	−	NE
Negative (vehicle) control [300]	3h / 20h	100	−	2.3
700 µg/mL [300]	3h / 20h	44	−^a	3.0
500 µg/mL [300]	3h / 20h	81	−^a	3.3
250 µg/mL [300]	3h / 20h	98	−^a	1.7
125 µg/mL	3h / 20h	98	−^a	NE
62.5 µg/mL	3h / 20h	110	−^a	NE
Positive control [73]	3h / 28h	64	−	68.5***

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 medium

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

 

Conclusions:

According to OECD 473 test method, CJ306 induced a significant level of chromosome aberrations in Chinese hamster V79 cells in the 20-hour treatment without metabolic activation.

Executive summary:

This test using the procedures outlined in the CiToxLAB Study Plan for 16/378-020C and OECD 473 (OECD, 2016).The results of this OECD 473 test for CJ306 show that test validity criteria was met.

Based on the cytotoxicity result, five concentrations of 700, 500, 250, 125 and 62.5 were used for Chromosome Aberration Assay 1 in the presence of S9-mix and five concentrations of 1250, 1000, 500, 250 and 125 μg/mL were used for Chromosome Aberration Assay 1 in the absence of S9-mix. Five concentrations of 62.5, 125, 250, 500 and 700µg/mL were used for Chromosome Aberration Assay 2 (3-hour treatment in the presence and 20-hour treatment in absence of S9-mix). None of the treatment concentrations caused a biologically or statistically significant increase in the number of cells with structural chromosome aberrations in Assay 1 with or without metabolic activation and in Assay 2 with metabolic activation when compared to the appropriate negative (vehicle) control values. However, in Assay 2 without metabolic activation, all 3 evaluated concentrations gave significant or highly significant increases in structural chromosome aberrations. Moreover, dose response was observed across all 3 doses.

In conclusion, no clastogenic activity was observed in case of the 3-hour treatments with and without metabolic activation. However, CJ306 induced a significant level of chromosome aberrations in Chinese hamster V79 cells in the 20-hour treatment without metabolic activation. Therefore, CJ306 was considered as clastogenic in this test system.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From March 9, 2020 to September 1, 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Vehicle / solvent:

RPMI1640

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Table 1. Statistic data of the treated cultures

Treatment condition	Doselevel/Control (μg/ml)	PE (%)	RPE (%)	SG	RSG (%)	RTG (%)	MF (×10^-6)	IMF (×10^-6)	MFsc (×10^-6)	IMFsc (×10^-6)
3h without S9	RPMI1640	93	100	13	100	100	119	0	103	0
	51.2	92	99	13	101	101	127	8	－	－
	128	78	85	12	95	81	147	28	－	－
	320	78	84	13	100	84	143	24	－	－
	800	77	83	12	89	74	167	48	－	－
	2000	88	95	10	78	74	129	10	116	13
	MMS(5.0)	64	69	12	90	62	528	409	459	355
3h with S9	RPMI1640	100	100	15	100	100	136	0	114	0
	51.2	102	102	14	92	95	134	-2	－	－
	128	93	93	14	89	83	144	8	－	－
	320	100	100	14	89	89	125	-11	－	－
	800	102	102	13	85	87	123	-13	－	－
	2000	107	107	12	80	86	133	-2	111	-3
	CP(3.0)	65	65	11	70	46	605	470	510	396
24h without S9	RPMI1640	93	100	66	100	100	109	0	98	0
	51.2	100	108	63	95	103	112	4	－	－
	128	92	100	64	97	96	132	23	－	－
	320	91	98	37	56	55	125	16	－	－
	800	88	95	13	20	19	146	37	116	18
	2000	63	68	6	9	6	－	－	－	－
	MMS(5.0)	71	76	37	56	43	606	498	462	363

Note: All results were the mean of duplicate cultures.

－: Not measured, not assessed.

Conclusions:

According to OECD 490 test method, CJ306 was negative effect under the condition of in vitro mammalian cell gene mutation test.

Executive summary:

This test using the procedures outlined in the SYRICI Study for G1982A0050 which is based on OECD 490 (OECD, 2016). The results of this OECD 490 test for CJ306 show that test validity criteria was met.

Based on cytotoxicity, 2000 μg/mL was set as the highest dose in this study. In the gene mutation test, five doses of CJ306 at 41.2, 128, 320, 800 and 2000 μg/mL, solvent and positive controls were tested in duplicate with or without metabolic activation. The IMF(s) were less than GEF at all analyzable designed doses in each treatment condition. Based on the data obtained from this study, it was concluded that under the test condition, CJ306 was negative effect in mammalian cell gene mutation test (in vitro).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

CJ306 was negative effect under the condition of in vivo mammalian somatic cell-erythrocyte micronucleus test (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 February 27, 2020 to September 1, 2020

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/female

Details on test animals or test system and environmental conditions:

- Source: SPF (Beijing) biotechnology Co., Ltd.
- Age at study initiation: 51-61 days old
- Weight at study initiation: Males: 35.50-38.87 g; Females: 29.12-31.83 g
- Housing: The male mice were housed one per cage and the female mice were housed 3 or 5 per cage.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days
- Temperature (°C): 21.7-23.6 °C
- Humidity (%): 56-62 %
- Photoperiod: 12-hrs dark / 12-hrs light

Route of administration:

intravenous

Vehicle:

Ultra-pure water (CAS No.: 7732-18-5)

Frequency of treatment:

Mice were administered twice by tail vein injection, with an interval of approximately 24 hours.

Dose / conc.:

25 mg/kg bw/day (nominal)

Remarks:

for females

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

for males and females

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

for males and females

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

for males

No. of animals per sex per dose:

five males and five females

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide monohydrate (CP, CAS No.: 6055-19-2)

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Table 1. Statistics results of the body weights in male and female mice

Sex	Group	Dose (mg/kg.bw)	Animal number	First Dosing (g) Mean±SD	Last Dosing (g) Mean±SD	Sacrifice (g) Mean±SD
Male	Negative control	0	1000-1004	37.09 ± 0.98	36.08 ± 0.40	36.78 ± 0.57
	Treated Group	50	1100-1104	37.22 ± 0.91	36.31 ± 1.97	37.81^*± 0.55
		100	1200-1204	37.23 ± 0.83	37.02 ± 0.97	36.90 ± 0.95
		200	1300-1304	37.30 ± 0.88	37.34 ± 0.36	37.01 ± 1.84
	CP	50	1400-1404	37.31 ± 1.07	36.01 ± 0.93	36.36 ± 1.44
Female	Negative control	0	2000-2004	30.35 ± 0.79	29.52 ± 1.12	30.06 ± 1.39
	Treated Group	25	2100-2104	30.47 ± 0.64	29.80 ± 0.71	28.75 ± 0.95
		50	2200-2204	30.48 ± 0.69	29.48 ± 1.23	29.83 ± 1.10
		100	2300-2304	30.48 ± 0.75	29.84 ± 0.70	29.59 ± 1.35
	CP	50	2400-2404	30.52 ± 0.84	29.14 ± 1.25	29.41 ± 1.47

Note: Statistically significant difference compared to negative control (^*: P<0.05).

 

Table 2. Statistics results of microscopic analysis in mice

Sex	Group	Animal number	Number of PCE	MNPCE/PCE (‰) Mean±SD	PCE/RBC Mean±SD
Male	Negative control	1000-1004	20000	1.7± 0.7	0.60 ± 0.05
	50	1100-1104	20000	1.7 ± 0.6	0.56^#± 0.03
	100	1200-1204	20000	1.8 ± 0.8	0.57^#± 0.04
	200	1300-1304	20000	1.6 ± 0.7	0.57^#± 0.10
	CP (50)	1400-1404	20000	25.6^**± 3.2	0.58 ± 0.05
Female	Negative control	2000-2004	20000	1.8 ± 0.6	0.60 ± 0.02
	25	2100-2104	20000	1.6 ± 0.6	0.57^#± 0.09
	50	2200-2204	20000	1.6 ± 0.7	0.56^#± 0.09
	100	2300-2304	20000	1.7 ± 0.4	0.54^#± 0.07
	CP (50)	2400-2404	20000	25.0^**± 3.5	0.55 ± 0.05

Note: Statistically significant difference compared to negative control (^**: P<0.01).

^#: the ratio was more than 20 percent of the ratio in the negative control group.

Conclusions:

According to OECD 474 test method, CJ306 was negative effect under the condition of in vivo mammalian somatic cell-erythrocyte micronucleus test.

Executive summary:

This test using the procedures outlined in the SYRICI Study for G1980C0060 which is based on OECD 474 (OECD, 2016). The results of this OECD 474 test for CJ306 show that test validity criteria was met.

According to the results of the preliminary test, the maximum tolerated dose (MTD) for male mice were 200 mg/kg.bw/day and for female mice were 100 mg/kg.bw/day. Therefore, the three dose levels in female mice were 25, 50 and 100 mg/kg.bw/day, and the three dose levels in the male mice were 50, 100 and 200 mg/kg.bw/day. Comparing with the concurrent negative control group, the incidence of micronucleated PCE for the treated groups had no ststistically significant difference (P>0.05) and the incidence of micronucleated PCE for CP group had ststistically significant difference (P<0.01). Furthermoer, the ratios betweeen PCE and RBC in all treated groups were more than 20 percent of the ratio in the concurrent negative control group. Under the conditions of this study, the results of micronucleus test were negative. Therefore, CJ306 was not to induce the increase of the incidence of micronucleated PCE in mice.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genetic toxicity in vitro

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 CJ306 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 5000μg/plate in the absence and presence of metabolite activations. Results showed that CJ306 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, CJ306 was not mutagenic in the reverse mutation analysis of Salmonellatyphimurium up to 5000μg/plate in the absence and presence of S9 metabolic activation.

 

Mammalian chromosomal aberration test (OECD TG473)

Based on the cytotoxicity result, five concentrations of 700, 500, 250, 125 and 62.5 were used for Chromosome Aberration Assay 1 in the presence of S9-mix and five concentrations of 1250, 1000, 500, 250 and 125 μg/mL were used for Chromosome Aberration Assay 1 in the absence of S9-mix. Five concentrations of 62.5, 125, 250, 500 and 700µg/mL were used for Chromosome Aberration Assay 2 (3-hour treatment in the presence and 20-hour treatment in absence of S9-mix). None of the treatment concentrations caused a biologically or statistically significant increase in the number of cells with structural chromosome aberrations in Assay 1 with or without metabolic activation and in Assay 2 with metabolic activation when compared to the appropriate negative (vehicle) control values. However, in Assay 2 without metabolic activation, all 3 evaluated concentrations gave significant or highly significant increases in structural chromosome aberrations. Moreover, dose response was observed across all 3 doses.

In conclusion, no clastogenic activity was observed in case of the 3-hour treatments with and without metabolic activation. However, CJ306 induced a significant level of chromosome aberrations in Chinese hamster V79 cells in the 20-hour treatment without metabolic activation. Therefore, CJ306 was considered as clastogenic in this test system.

Mammalian cell gene mutation test(OECD TG490)

Based on cytotoxicity, 2000 μg/mL was set as the highest dose in this study. In the gene mutation test, five doses of CJ306 at 41.2, 128, 320, 800 and 2000 μg/mL, solvent and positive controls were tested in duplicate with or without metabolic activation. The IMF(s) were less than GEF at all analyzable designed doses in each treatment condition. Based on the data obtained from this study, it was concluded that under the test condition, CJ306 was negative effect in mammalian cell gene mutation test (in vitro).

 

Genetic toxicity in vivo

Mammalian erythrocyte micronucleus test(OECD TG474)

According to the results of the preliminary test, the maximum tolerated dose (MTD) for male mice were 200 mg/kg.bw/day and for female mice were 100 mg/kg.bw/day. Therefore, the three dose levels in female mice were 25, 50 and 100 mg/kg.bw/day, and the three dose levels in the male mice were 50, 100 and 200 mg/kg.bw/day. Comparing with the concurrent negative control group, the incidence of micronucleated PCE for the treated groups had no ststistically significant difference (P>0.05) and the incidence of micronucleated PCE for CP group had ststistically significant difference (P<0.01). Furthermoer, the ratios betweeen PCE and RBC in all treated groups were more than 20 percent of the ratio in the concurrent negative control group. Under the conditions of this study, the results of micronucleus test were negative. Therefore, CJ306 was not to induce the increase of the incidence of micronucleated PCE in mice.