5-({4-chloro-6-[ethyl(phenyl)amino]-1,3,5-triazin-2-yl}amino)-4-hydroxy-3-[(2-sulfophenyl)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

CJ305 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(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 January 17, 2017 to April 6, 2017

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:

S9 mix activation

Untreated negative controls:

yes

Remarks:

sterile deionized water

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

congo red

mitomycin C

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

Evaluation criteria:

Acceptable ranges of background revertants from the mean value of triplicateplates for five tester strains without S9 metabolic activation 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 Test 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	+	+	+	+	+
ΔuvrBmutation	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 CJ305 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	30	23	26	67	85	70	365	385	378	22	15	17	19	14	14
	M^f	26 ± 4			74 ± 10			376 ± 10			18 ± 4			16 ± 3
50	I^e	36	20	20	52	74	60	394	384	357	16	17	17	20	19	18
	M^f	25 ± 9			62 ± 11			378 ± 19			17 ± 1			19 ± 1
150	I^e	35	23	13	57	68	71	397	367	362	12	19	21	12	19	14
	M^f	24 ± 11			65 ± 7			375 ± 19			17 ± 5			15 ± 4
500	I^e	15	24	35	65	69	75	378	339	370	12	11	17	10	17	22
	M^f	25 ± 10			70 ± 5			362 ± 21			13 ± 3			16 ± 6
1500	I^e	16	18	20	73	73	80	350	378	384	14	12	17	15	16	23
	M^f	18 ± 2			75 ± 4			371 ± 18			14 ± 3			18 ± 4
5000	I^e	19	10	19	74	55	71	380	332	393	28	18	16	20	23	22
	M^f	16 ± 5			67 ± 10			367 ± 32			21 ± 6			22 ± 2
Positive control^b	I^e	193	254	214	395	438	416	2292	1890	1870	379	352	304	58	52	40
	M^f	220^c± 31			416^c±22			2017^c± 238			345^d± 38			50^d± 9

a: Negative control was sterile deionized water.

b: Positive controls: 1 μg/plate 2-nitrofluorene for TA98

0.5 μg/plate sodium azide for TA100

62.5 ng/plate mitomycin C for TA102

0.1 μg/plate sodium azide for TA1535

0.3 μg/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 C305 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	43	39	52	90	105	93	495	270	626	14	10	11	35	25	29
	M^f	45 ± 7			96 ± 8			464 ± 180			12 ± 2			30 ± 5
50	I^e	39	38	35	82	84	91	406	555	527	11	16	14	19	34	8
	M^f	37 ± 2			86 ± 5			496 ± 79			14 ± 3			20 ± 13
150	I^e	45	35	38	92	89	99	506	213	427	10	13	12	31	22	16
	M^f	39 ± 5			93 ± 5			382 ± 152			12 ± 2			23 ± 8
500	I^e	33	35	31	106	92	93	492	492	471	14	8	14	28	34	19
	M^f	33 ± 2			97 ± 8			485 ± 12			12 ± 3			27 ± 8
1500	I^e	31	40	39	99	83	71	561	506	486	10	17	12	23	28	24
	M^f	37 ± 5			84 ± 14			518 ± 39			13 ± 4			25 ± 3
5000	I^e	29	23	26	61	90	89	407	475	452	10	14	13	16	23	24
	M^f	26 ± 3			80 ± 16			445 ± 35			12 ± 2			21 ± 4
Positive control^b	I^e	376	310	301	314	351	347	1570	1152	1362	406	315	300	387	309	240
	M^f	329^c± 41			337^c± 20			1361^c± 209			340^d± 57			312^d± 74

a: Negative control was sterile deionized water.

b: Positive controls: 60 μg/plate Congo Red for TA98

1 μg/plate 2-aminofluorene for TA100

2 μg/plate 2-aminoanthracene for TA102

0.5 μg/plate 2-aminoanthracene for TA1535

2 μg/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, CJ305 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 T65316018-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 CJ305 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 CJ305 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 CJ305 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, CJ305 was not mutagenic in the reverse mutation analysis of Salmonellatyphimurium up to 5000 μg/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, 5000 μg/plate was set as the highest dose in this study. In the mutagenicity assay, five doses of CJ305 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 CJ305 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, CJ305 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.