7-[{4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-2-(carbamoylamino)phenyl}diazenyl]naphthalene-1,3,6-trisulfonic acid, lithium sodium salts

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

CJ312 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).

Link to relevant study records

Reference

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 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

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:

no cytotoxicity

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:

no cytotoxicity

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:

no cytotoxicity

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:

no cytotoxicity

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:

no cytotoxicity

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 CJ312 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	19	23	25	81	84	55	335	394	407	6	13	20	14	14	16
	M^f	22 ± 3			73 ± 16			379 ± 38			13 ± 7			15 ± 1
50	I^e	22	28	25	89	84	63	360	352	366	24	24	13	24	17	18
	M^f	25 ± 3			79 ± 14			359 ± 7			20 ± 6			20 ± 4
150	I^e	18	17	14	82	59	66	382	407	423	12	10	13	17	16	6
	M^f	16 ± 2			69 ± 12			404 ± 21			12 ± 2			13 ± 6
500	I^e	25	27	30	73	72	83	372	358	379	12	13	21	11	14	12
	M^f	27 ± 3			76 ± 6			370 ± 11			15 ± 5			12 ± 2
1500	I^e	30	28	13	89	72	65	360	387	380	16	19	22	14	8	18
	M^f	24 ± 9			75 ± 12			376 ± 14			19 ± 3			13 ± 5
5000	I^e	21	24	25	87	79	72	329	351	360	21	17	11	16	16	20
	M^f	23 ± 2			79±8			347 ± 16			16 ± 5			17 ± 2
Positive control^b	I^e	229	249	258	579	566	533	1450	1254	1272	419	366	321	167	199	223
	M^f	245^c± 15			559^c± 24			1325^c± 108			369^d± 49			196^d± 28

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 CJ312 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	34	32	34	88	122	78	338	336	376	7	13	12	14	19	10
	M^f	33 ± 1			96 ± 23			350 ± 23			11 ± 3			14 ± 5
50	I^e	47	15	34	90	93	98	338	369	367	7	7	15	11	14	15
	M^f	32 ± 16			94 ± 4			358 ± 17			10 ± 5			13 ± 2
150	I^e	29	26	29	95	76	101	366	338	382	10	17	11	19	14	13
	M^f	28 ± 2			91 ± 13			362 ± 22			13 ± 4			15 ± 3
500	I^e	29	20	35	91	78	82	310	322	359	7	12	15	6	20	9
	M^f	28 ± 8			84 ± 7			330 ± 26			11 ± 4			12 ± 7
1500	I^e	29	19	31	67	90	94	357	339	352	17	3	11	12	10	14
	M^f	26 ± 6			84 ± 15			349 ± 9			10 ± 7			12 ± 2
5000	I^e	27	16	26	105	97	84	401	396	335	9	16	10	9	12	13
	M^f	23 ± 6			95 ± 11			377 ± 37			12 ± 4			11 ± 2
Positive control^b	I^e	177	193	191	663	708	641	1822	1914	1274	273	261	252	286	275	306
	M^f	187^c± 9			671^c± 34			1670^c± 346			262^d± 11			289^d± 16

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, CJ312 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 T65315030-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 CJ312 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 CJ312 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 CJ312 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, CJ312 was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 5000μg/plate in the absence and presence of S9 metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genetic toxicity in vitro

Based on the preliminary assay results, 5000μg/plate was set as the highest dose in this study. In the mutagenicity assay, five doses of CJ312 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 CJ312 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, CJ312 was not mutagenic in the reverse mutation analysis ofSalmonellatyphimuriumup to 5000μg/plate in the absence and presence of S9 metabolic activation.