2-[{6-[(4-amino-6-chloro-1,3,5-triazin-2-yl)(methyl)amino]-1-hydroxy-3-sulfonaphthalen-2-yl}diazenyl]naphthalene-1,5-disulfonic acid, lithium sodium salts

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

CJ302 was not mutagenic in the reverse mutation analysis of Salmonella typhimuriumup to 5000 μg/plate in the absence and presence of S9 metabolic activation (OECD TG471).

CJ302 did not induce chromosome aberration in CHO cells in absence or presence of S9 metabolic activation (OECD TG473).

CJ302 was negative effect under the condition of in vitro mammalian cell gene mutation test (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 October 22, 2015 to June 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

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	+	+	+	+	+
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 CJ302 inSalmonella typhimuriumStrains 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	29	37	43	97	74	89	313	395	326	23	18	17	15	11	9
	M^f	36 ± 7			87 ± 12			345 ± 44			19 ± 3			12 ± 3
50	I^e	39	37	47	78	78	72	355	348	355	15	21	25	15	8	12
	M^f	41 ± 5			76 ± 3			353 ± 4			20 ± 5			12 ± 4
150	I^e	39	48	45	81	87	77	298	344	400	22	27	26	10	12	8
	M^f	44 ± 5			82 ± 5			347 ± 51			25 ± 3			10 ± 2
500	I^e	49	30	43	90	68	87	235	359	297	13	21	20	6	8	10
	M^f	41 ± 10			82 ± 12			297 ± 62			18 ± 4			8 ± 2
1500	I^e	41	33	31	77	76	99	268	310	369	27	18	27	8	6	13
	M^f	35 ± 5			84 ± 13			316 ± 51			24 ± 5			9 ± 4
5000	I^e	23	34	42	86	61	72	338	343	364	17	19	22	9	9	13
	M^f	33 ± 10			73±13			348 ± 14			19 ± 3			10 ± 2
Positive control^b	I^e	310	221	252	529	546	545	1069	975	1012	339	336	331	302	380	351
	M^f	261^c± 45			540^c± 10			1019^c± 47			345^d± 18			344^d± 39

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 CJ302 in Salmonella typhimurium Strains with 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	27	29	27	106	122	80	388	357	366	16	13	18	13	18	11
	M^f	28 ± 1			103 ± 21			370 ± 16			16 ± 3			14 ± 4
50	I^e	39	22	32	120	71	145	323	333	360	16	14	9	14	10	8
	M^f	31 ± 9			112 ± 38			339 ± 19			13 ± 4			11 ± 3
150	I^e	29	35	34	97	60	83	349	318	325	16	18	9	10	19	24
	M^f	33 ± 3			80 ± 19			331 ± 16			14 ± 5			18 ± 7
500	I^e	20	34	22	77	152	122	245	354	298	14	13	12	10	17	13
	M^f	25 ± 8			117 ± 38			299 ± 55			13 ± 1			13 ± 4
1500	I^e	28	37	39	101	124	88	361	345	350	17	14	16	14	8	7
	M^f	35 ± 6			104 ± 18			352 ± 8			16 ± 2			10 ± 4
5000	I^e	37	27	26	128	100	125	275	362	314	24	16	16	13	14	15
	M^f	30 ± 6			118 ± 15			317 ± 44			19 ± 5			14 ± 1
Positive control^b	I^e	162	201	173	655	711	668	975	930	1274	203	213	201	283	323	334
	M^f	179^c± 20			678^c± 29			1060^c± 187			206^d± 6			313^d± 27

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, CJ302 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 T65315026-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 CJ302 show that test validity criteria was met.

Based on the preliminary assay results, 5000 μg/platewas set as the highest dose in this study. In the mutagenicity assay, five doses of CJ302 at 50, 150, 500, 1500 and 5000 μg/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 5000 μg/plate in the absence and presence of metabolite activations. Results showed that CJ302 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, CJ302 was not mutagenic in the reverse mutation analysis of Salmonella typhimuriumup to 5000 μg/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 November 08, 2016 to May 16, 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	1	20	25	4	0	0

Table 2. Concurrent Cytotoxicity Analysis of CJ302 in Chinese Hamster Ovary Cells

Concentration (µg/mL)	Cell Number (× 10^5cells)	ICC^a N-N0	RICC^b (%)	Cytotoxicity^c (%)
Before Treatment
Untreated (-9S)	21.4
Untreated (+9S)	28.0
After Treatment
Scheme I (-S9, 3h)
Negative Control	61.6	40.2	100.0	0.0
51.2	56.8	35.4	88.1	11.9
128	57.8	36.4	90.5	9.5
320	54.6	33.2	82.6	17.4
800	55.2	33.8	84.1	15.9
2000	53.8	32.4	80.6	19.4
Positive Control^d	47.0	25.6	63.7	36.3
Scheme II (+S9, 3h)
Negative Control	58.2	30.2	100.0	0.0
51.2	74.2	46.2	153.0	0.0
128	52.4	24.4	80.8	19.2
320	71.8	43.8	145.0	0.0
800	55.0	27.0	89.4	10.6
2000	56.2	28.2	93.4	6.6
Positive Control^e	49.0	21.0	69.5	30.5
Scheme I (-S9, 20h)
Negative Control	56.2	34.8	100.0	0.0
51.2	57.0	35.6	102.3	0.0
128	54.6	33.2	95.4	4.6
320	57.8	36.4	104.6	0.0
800	91.8	70.4	202.3	0.0
2000	47.2	25.8	74.1	25.9
Positive Control^f	61.4	40.0	114.9	0.0

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 CJ302

Treatment	Concentration (µg/mL)	Treating Hour	S9 (-/+)	Aberrant Cells (%)
Scheme I (-S9, 3h)
Negative Control	0	3	-	0.00
Test Article	51.2	3	-	0.00
	128	3	-	0.00
	320	3	-	1.00
	800	3	-	0.67
	2000	3	-	0.67
Positive Control(MMC)	0.33	3	-	21.00*
Scheme II (+S9, 3h)
Negative Control	0	3	+	0.00
Test Article	51.2	3	+	0.00
	128	3	+	0.00
	320	3	+	0.33
	800	3	+	0.00
	2000	3	+	1.00
Positive Control (CPP)	11.2	3	+	22.67*
Scheme I (-S9, 20h)
Negative Control	0	20	-	0.00
Test Article	51.2	20	-	0.00
	128	20	-	0.67
	320	20	-	1.00
	800	20	-	1.00
	2000	20	-	0.33
Positive Control(MMC)	0.2	20	-	32.00*

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

Conclusions:

According to OECD 473 test method, CJ302 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 Study Plan for T65316026-GT and OECD 473 (OECD, 2016). The results of this OECD 473 test for CJ302 show that test validity criteria was met.

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 assay. All tests were conducted in duplicate with concurrent negative and positive controls. Results showed that the percentage of aberrant cells of negative controls in all three test schemes was 0%. The positive controls induced significant increases in percentages (21.00%, 22.67% and 32.00%, respectively) of aberrant cells as compared to the corresponding negative control. There were at least three analyzable concentrations obtained for each test scheme, which met the requirements for a valid test. In all three schemes, the test article treated cell cultures did not show more than 3% of the frequencies of structural chromosome aberration. Therefore,CJ302 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:

experimental study

Adequacy of study:

key study

Study period:

From October 15, 2018 to January 2, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test) (migrated information)

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:

It was no cytotoxic effect (the cell survival rate was >50 %) in 2 mg/mL with and without S9 Mix.

True negative controls:

yes

Remarks:

Ham's F-12

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

CHO-K1

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

at the highest dosage (2.0 mg/mL)

Positive controls validity:

valid

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^c	60.0 ± 7.1	100.00 ± 0.00
	Positive control^d	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

^aValues were expressed as Mean ± S.D., and tests were repeated two times.

^bRelative survival = each colony numbers of the positive control or test groups / the average of colony numbers in the negative control × 100%, then calculated the Mean ± S.D..

^cNegative control: Ham’s F-12 medium with 10% FBS (S9 Mix or not).

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

 

Table 2. Mutation frequency analysis

Group	Test article	Average colony numbers^a	Mutation frequency (× 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^c	15..3 ± 7.4	17.1
	Positive control^d	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

^aValues were expressed as Mean ± S.D., and tests were repeated three times.

^bMutation frequency = (numbers of colonies /number of seeding) × (1 / Colonies formation frequency).

^cNegative control: Ham’s F-12 medium with 10% FBS (S9 Mix or not).

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

* Significantly different from the negative control group (ρ < 0.005).

Conclusions:

According to OECD 476 test method, CJ302 was negative effect under the condition of in vitro mammalian cell gene mutation test.

Executive summary:

This test using the procedures outlined in the SuperLub Study Plan for M62-180800004001EN which is based on the SOP for the OECD 476 (SOPF-240) and OECD 476 (OECD, 2015).The results of this OECD 476 test for CJ302 show that test validity criteria was met.

Based on the results of the cell viability test, 2.0mg/mL was set as the highest dose in this study. In the gene mutation test, four doses of CJ302 at 0.25, 0.5, 1.0 and 2.0mg/mL, negative and positive controls were tested infive repetitionswith or without S9 Mix. The mutation frequency was no significantly different from the negative control group for all test groups in the absence and presence of S9 Mix. Based on the data obtained from this study, it was concluded that under the test condition, CJ302 was negative effect in mammalian cell gene mutation test (in vitro).

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, 5000 μg/plate was set as the highest dose in this study. In the mutagenicity assay, five doses of CJ302 at 50, 150, 500, 1500 and 5000 μg/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 CJ302 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, CJ302 was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 5000 μg/plate in the absence and presence of S9 metabolic activation.

Mammalian chromosomal aberration test (OECD TG473)

Based on the cytotoxicity result, five concentrations of 51.2, 128, 320, 800 and 2000µg/mL were used for all three schemes in the assay. All tests were conducted in duplicate with concurrent negative and positive controls. Results showed that the percentage of aberrant cells of negative controls in all three test schemes was 0%. The positive controls induced significant increases in percentages (21.00%, 22.67% and 32.00%, respectively) of aberrant cells as compared to the corresponding negative control. There were at least three analyzable concentrations obtained for each test scheme, which met the requirements for a valid test. In all three schemes, the test article treated cell cultures did not show more than 3% of the frequencies of structural chromosome aberration. Therefore, CJ302 did not induce chromosome aberration in CHO cells in absence or presence of S9 metabolic activation.

Mammalian cell gene mutation tests (OECD TG476)

Based on the results of the cell viability test, 2.0mg/mL was set as the highest dose in this study. In the gene mutation test, four doses of CJ302 at 0.25, 0.5, 1.0 and 2.0mg/mL, negative and positive controls were tested infive repetitionswith or without S9 Mix. The mutation frequency was no significantly different from the negative control group for all test groups in the absence and presence of S9 Mix. Based on the data obtained from this study, it was concluded that under the test condition, CJ302 was negative effect in mammalian cell gene mutation test (in vitro).