4-[(4-chloro-6-{[(3 or 4)-sulfophenyl]amino}-1,3,5-triazin-2-yl)amino]-2-{[1-ethyl-2-hydroxy-4-methyl-6-oxo-5-(sulfonatomethyl)-1,6-dihydropyridin-3-yl]diazenyl}benzenesulfonic acid, lithium sodium salts

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance 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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to 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) from Aroclor 1254-induced Sprague-Dawley rats

Vehicle / solvent:

DMSO was used as the vehicle for positive controls.

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 CJ301 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	34	19	28	99	82	79	457	461	537	12	25	17	11	12	12
	M^f	27 ± 8			87 ± 11			485 ± 45			18 ± 7			12 ± 1
50	I^e	30	28	30	87	94	85	477	461	487	15	25	22	10	19	9
	M^f	29 ± 1			89 ± 5			475 ± 13			21 ± 5			13 ± 6
150	I^e	31	24	25	85	99	84	531	497	519	16	15	24	18	10	17
	M^f	27 ± 4			89 ± 8			516 ± 17			18 ± 5			15 ± 4
500	I^e	30	28	23	74	89	92	527	517	495	16	18	24	13	14	15
	M^f	27 ± 4			84 ± 10			513 ± 16			19 ± 4			14 ± 1
1500	I^e	33	36	30	96	96	80	445	509	509	16	21	18	14	18	20
	M^f	33 ± 3			91 ± 9			488 ± 37			18 ± 3			17 ± 3
5000	I^e	36	21	24	87	92	86	511	529	453	23	22	29	10	15	12
	M^f	27 ± 8			88±3			498 ± 40			25 ± 4			12 ± 3
Positive control^b	I^e	264	277	301	596	568	568	1550	1310	1551	378	337	351	373	351	390
	M^f	281^c± 19			577^c± 16			1470^c± 139			355^d± 21			371^d± 20

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 CJ301 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	31	33	35	92	78	87	327	332	379	11	11	7	8	13	16
	M^f	33 ± 2			86 ± 7			346 ± 29			10 ± 2			12 ± 4
50	I^e	41	22	32	91	87	98	326	439	280	9	12	14	11	10	23
	M^f	32 ± 10			92 ± 6			348 ± 82			12 ± 3			15 ± 7
150	I^e	28	39	33	94	85	94	209	299	265	9	12	14	17	16	24
	M^f	33 ± 6			91 ± 5			258 ± 45			12 ± 3			19 ± 4
500	I^e	29	31	29	79	99	91	311	375	321	13	7	15	15	17	16
	M^f	30 ± 1			90 ± 10			336 ± 34			12 ± 4			16 ± 1
1500	I^e	32	19	30	75	106	92	405	404	232	9	9	5	8	14	13
	M^f	27 ± 7			91 ± 16			347 ± 100			8 ± 2			12 ± 3
5000	I^e	24	29	32	87	82	98	323	417	349	7	11	12	16	13	8
	M^f	28 ± 4			89 ± 8			363 ± 49			10 ± 3			12 ± 4
Positive control^b	I^e	219	239	214	539	602	497	1386	1708	1900	226	232	240	294	245	301
	M^f	224^c± 13			546^c± 53			1665^c± 260			233^d± 7			280^d± 31

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, CJ301 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 T65315025-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 CJ301 show that test validity criteria was met.

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 CJ301 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 CJ301 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, CJ301 was not mutagenic in the reverse mutation analysis of Salmonella typhimuriumup to 5000 μg/plate in the absence and presence of S9 metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

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 CJ301 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 CJ301 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, CJ301 was not mutagenic in the reverse mutation analysis of Salmonella typhimuriumup to 5000 μg/plate in the absence and presence of S9 metabolic activation.