5-({4-chloro-6-[ethyl(phenyl)amino]-1,3,5-triazin-2-yl}amino)-4-hydroxy-3-[(1-sulfo-2-naphthyl)diazenyl]naphthalene-2,7-disulfonic 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 April 20, 2016 to October 14, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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:

5-({4-chloro-6-[ethyl(phenyl)amino]-1,3,5-triazin-2-yl}amino)-4-hydroxy-3-[(1-sulfo-2-naphthyl)diazenyl]naphthalene-2,7-disulfonic acid, lithium sodium salts

EC Number:

942-803-7

Molecular formula:

Not applicable; this UVCB substance contains: C31H21ClN7O10S3.xLi.yNa, (x + y) = 3; 0 < (x,y) < 3 with 804.0 < MW < 852.1 g/mol (UVCB substance), C31H22N7O11S3.xLi.yNa, (x + y) = 3; 0 < (x,y) < 3 with 785.5 < MW < 833.7 g/mol (UVCB substnace), and traces of NaCl and Na2SO4.

IUPAC Name:

5-({4-chloro-6-[ethyl(phenyl)amino]-1,3,5-triazin-2-yl}amino)-4-hydroxy-3-[(1-sulfo-2-naphthyl)diazenyl]naphthalene-2,7-disulfonic 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 Aroclor 1254-induced Sprague-Dawley rats

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

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:

not specified

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:

not specified

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:

not specified

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:

not specified

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:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Table 1. Genotype Confirmation Tests of Salmonella typhimuriumTester 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 CJ306 in Salmonella typhimurium Strains 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	13	41	17	62	80	65	326	312	374	19	10	13	8	9	6
	M^f	24 ± 15			69 ± 10			337 ± 33			14 ± 5			8 ± 2
50	I^e	25	41	38	66	67	54	317	247	274	13	11	12	12	6	4
	M^f	35 ± 9			62 ± 7			279 ± 35			12 ± 1			7 ± 4
150	I^e	29	32	20	83	80	64	218	274	400	11	12	19	10	4	6
	M^f	27 ± 6			76 ± 10			297 ± 93			14 ± 4			7 ± 3
500	I^e	19	27	22	62	67	68	257	309	302	20	17	15	4	11	12
	M^f	23 ± 4			66 ± 3			289 ± 28			17 ± 3			9 ± 4
1500	I^e	22	30	25	69	50	72	286	247	191	9	8	8	10	10	8
	M^f	26 ± 4			64 ± 12			241 ± 48			8 ± 1			9 ± 1
5000	I^e	21	27	24	66	50	68	249	272	251	9	15	13	9	6	5
	M^f	24 ± 3			61 ± 10			257 ± 13			12 ± 3			7 ± 2
Positive control^b	I^e	190	220	205	584	483	589	1061	1259	1060	370	425	410	78	90	110
	M^f	205^c± 15			552^c± 60			1127^c± 115			402^d± 28			93^d± 16

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 CJ306 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	21	18	29	89	78	90	453	461	416	11	13	9	10	5	10
	M^f	23 ± 6			86 ± 7			443 ± 24			11 ± 2			8 ± 3
50	I^e	23	16	12	103	82	87	385	418	385	9	6	9	6	7	12
	M^f	17 ± 6			91 ± 11			396 ± 19			8 ± 2			8 ± 3
150	I^e	26	20	21	98	81	80	409	396	431	9	8	7	9	9	14
	M^f	22 ± 3			86 ± 10			412 ± 18			8 ± 1			11 ± 3
500	I^e	23	22	22	85	91	89	391	392	408	14	14	12	16	10	4
	M^f	22 ± 1			88 ± 3			397 ± 10			13 ± 1			10 ± 6
1500	I^e	15	26	17	73	71	88	333	365	361	13	7	7	4	9	10
	M^f	19 ± 6			77 ± 9			353 ± 17			9 ± 3			8 ± 3
5000	I^e	17	24	23	74	71	84	329	308	306	13	13	9	6	11	7
	M^f	21 ± 4			76 ± 7			314 ± 13			12 ± 2			8 ± 3
Positive control^b	I^e	174	162	141	597	573	609	1968	2178	2076	152	167	180	205	183	209
	M^f	159^c± 17			593^c± 18			2074^c± 105			166^d± 14			199^d±14

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

Applicant's summary and conclusion

Conclusions:

According to OECD 471 test method, CJ306 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 T65315028-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 CJ306 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 CJ306 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 CJ306 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, CJ306 was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 5000μg/plate in the absence and presence of S9 metabolic activation.