2-{[4-chloro-6-({8-hydroxy-3,6-disulfo-7-[(1-sulfo-2-naphthyl)diazenyl]-1-naphthyl}amino)-1,3,5-triazin-2-yl]amino}-5-sulfobenzoic acid, lithium sodium salts

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From December 29, 2016 to June 13, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-{[4-chloro-6-({8-hydroxy-3,6-disulfo-7-[(1-sulfo-2-naphthyl)diazenyl]-1-naphthyl}amino)-1,3,5-triazin-2-yl]amino}-5-sulfobenzoic acid, lithium sodium salts

EC Number:

943-083-7

Cas Number:

118578-12-4

Molecular formula:

Not applicable; this UVCB substance contains: C30H15ClN7O15S4.xLi.yNa, (x + y) = 5; 0 < (x,y) < 5 with 911.8 < MW < 992.1 g/mol (UVCB substance), C30H16N7O16S4.xLi.yNa, (x + y) = 5; 0 < (x,y) < 5 with 893.4 < MW < 973.7 g/mol (UVCB substnace), C23H11Cl2N6O10S3.xLi.yNa, (x + y) = 3; 0 < (x,y) < 3 with 719.3 < MW < 767.4 g/mol (UVCB substnace), and traces of NaCl.

IUPAC Name:

2-{[4-chloro-6-({8-hydroxy-3,6-disulfo-7-[(1-sulfo-2-naphthyl)diazenyl]-1-naphthyl}amino)-1,3,5-triazin-2-yl]amino}-5-sulfobenzoic acid, lithium sodium salts

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Method

Species / strain

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 uninduced male Golden Syrian Hamster liver

Controls

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:

Tester Strain Revertants
TA98 10-60
TA100 50-240
TA102 180-480
TA1535 5-45
TA1537 2-25

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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

Any other information on results incl. tables

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 CJ308 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	20	33	61	98	79	271	270	341	15	17	19	11	13	13
	M^f	28 ± 7			79 ± 19			294 ± 41			17 ± 2			12 ± 1
50	I^e	13	21	19	66	82	68	305	319	328	17	20	18	17	5	10
	M^f	18 ± 4			72 ± 9			317 ± 12			18 ± 2			11 ± 6
150	I^e	17	18	16	72	71	61	256	270	334	14	23	18	15	5	11
	M^f	17 ± 1			68 ± 6			287 ± 42			18 ± 5			10 ± 5
500	I^e	25	15	18	68	92	60	275	241	300	11	17	9	7	12	7
	M^f	19±5			73 ± 17			272 ± 30			12 ± 4			9 ± 3
1500	I^e	16	23	21	51	91	60	250	265	319	12	11	13	9	6	8
	M^f	20 ± 4			67 ± 21			278 ± 36			12 ± 1			8 ± 2
5000	I^e	17	22	23	61	96	85	232	268	319	8	16	16	13	15	7
	M^f	21 ± 3			81 ± 18			273 ± 44			13 ± 5			12 ± 4
Positive control^b	I^e	266	275	238	421	481	509	1996	1962	2130	374	424	235	180	153	146
	M^f	260^c± 19			470^c± 45			2029^c± 89			344^d± 98			160^d± 18

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 CJ308 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	56	44	41	104	121	104	545	463	609	9	18	15	28	34	26
	M^f	47 ± 8			110 ± 10			539 ± 73			14 ± 5			29 ± 4
50	I^e	58	37	38	83	97	103	567	423	495	9	19	11	26	38	33
	M^f	44 ± 12			94 ± 10			495 ± 72			13 ± 5			32 ± 6
150	I^e	38	54	41	97	98	104	582	563	574	7	19	10	28	23	24
	M^f	44 ± 9			100 ± 4			573 ± 10			12 ± 6			25 ± 3
500	I^e	42	33	53	97	67	78	570	574	565	17	14	16	27	26	29
	M^f	43 ± 10			81 ± 15			570 ± 5			16 ± 2			27 ± 2
1500	I^e	36	31	25	79	99	96	590	410	526	11	7	15	29	25	20
	M^f	31 ± 6			91 ± 11			509 ± 91			11 ± 4			25 ± 5
5000	I^e	33	31	20	65	65	92	516	393	506	12	7	8	20	24	17
	M^f	28 ± 7			74 ± 16			472 ± 68			9 ± 3			20 ± 4
Positive control^b	I^e	277	226	189	299	320	343	1518	1644	1930	254	254	293	319	292	351
	M^f	231^c± 44			321^c± 22			1697^c± 211			267^d± 23			321^d± 30

a: Negative control was sterile deionized water.

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

1μg/plate 2-aminofluorene for TA100

2 μg/plate 2-aminoanthracene for TA102

0.5μg/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.

Applicant's summary and conclusion

Conclusions:

According to OECD 471 test method, CJ308 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 T65316019-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 CJ308 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 CJ308 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 toxicity was observed in all five tester strains up to 5000 μg/plate in the absence and presence of metabolite activations. Results showed that CJ308 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, CJ308 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.