2-{[4-({4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-5-sulfo-1-naphthyl}diazenyl)-(6 or 7)-sulfo-1-naphthyl]diazenyl}benzene-1,4-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 January 17, 2017 to July 03, 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:

Reaction products of 1,4-Benzenedisulfonic acid, 2-[[4-[[4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-5-sulfo-1-naphthalenyl]azo]sulfo-1-naphthalenyl]azo]-, tetrasodium salt and lithium chloride

Molecular formula:

Not applicable; this UVCB substance contains: C29H16ClN9O12S4.xLi.yNa, (x + y) = 4; 0 < (x,y) < 4 with 873.9 < MW < 938.1 g/mol (UVCB substance), and traces of NaCl.

IUPAC Name:

Reaction products of 1,4-Benzenedisulfonic acid, 2-[[4-[[4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-5-sulfo-1-naphthalenyl]azo]sulfo-1-naphthalenyl]azo]-, tetrasodium salt and lithium chloride

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-Amoinoanthracene

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 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 4

Species / strain:

S. typhimurium TA 102

Metabolic activation:

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 102

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 μg/plate

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 6

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 7

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 μg/plate

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The substance was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 1500 ug/plate.

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 CJ313 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	27	25	31	50	53	70	288	322	268	16	6	13	21	14	15
	M^f	28 ± 3			58 ± 11			293 ± 27			12 ± 5			17 ± 4
50	I^e	29	23	30	56	61	53	322	336	318	18	12	16	4^h	9	12
	M^f	27 ± 4			57 ± 4			325 ± 9			15 ± 3			8^g± 4
150	I^e	14	18	18	58	71	52	216	350	280	13	16	22	17	11	14
	M^f	17 ± 2			60 ± 10			282 ± 67			17 ± 5			14 ± 3
500	I^e	18	17	26	57	49	80	332	256	204	12	15	16	6	12	16
	M^f	20 ± 5			62 ± 16			261 ± 59			14 ± 2			11 ± 5
1500	I^e	23	25	23	70	61	58	294	294	278	14	17	16	17	10	9
	M^f	24 ± 1			63 ± 6			289 ± 9			16 ± 2			12 ± 4
5000	I^e	25	22	17	42	60	57	258	176	292	13	11	18	10	13	16
	M^f	21 ± 4			53 ± 10			242 ± 60			14 ± 4			13 ± 3
Positive control^b	I^e	181	255	266	374	374	430	1411	1229	1376	439	428	349	191	181	174
	M^f	234^c± 46			393^c±32			1339^c± 97			405^d± 49			182^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.

g: Cytotoxicty: a > 50% reduction when compared to the mean colony number of negative control revertants.

h: Extreme low data may consider as an outlier (out of range of historical data).

 

Table 3. Mutagenicity Test of CJ313 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	38	41	38	86	75	89	515	486	562	15	11	13	29	33	32
	M^f	39 ± 2			83 ± 7			521 ± 38			13 ± 2			31 ± 2
50	I^e	28	40	37	63	108	70	337	414	483	12	14	18	28	22	19
	M^f	35 ± 6			80 ± 24			411 ± 73			15 ± 3			23 ± 5
150	I^e	43	38	47	61	76	88	422	416	131	7	15	11	26	24	31
	M^f	43 ± 5			75 ± 14			384 ± 61			11 ± 4			27 ± 4
500	I^e	23	36	41	84	88	67	296	316	390	15	16	17	29	35	34
	M^f	33 ± 9			80 ± 11			334 ± 50			16 ± 1			33 ± 3
1500	I^e	36	35	33	93	77	90	294	367	391	12	12	11	18	18	13
	M^f	35 ± 2			87 ± 9			351 ± 51			12 ± 1			16 ± 3
5000	I^e	27	27	17	65	54	80	343	179	136	10	11	7	10	8	16
	M^f	24 ± 6			66 ± 13			219^g± 109			9 ± 2			11^g± 4
Positive control^b	I^e	263	329	312	254	298	346	1133	1336	1086	300	307	459	146	132	110
	M^f	301^c± 34			299^c± 46			1185^c± 133			355^d± 90			129^d± 18

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.

g: Cytotoxicty: a > 50% reduction when compared to the mean colony number of negative control revertants.

Applicant's summary and conclusion

Conclusions:

According to OECD 471 test method, CJ313 was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 1500 μg/plate.

Executive summary:

This test using the procedures outlined in the QPS Taiwan Study Plan for T65316020-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 CJ313 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 CJ313 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.Cytotoxicity was observed in tester strains TA102 and TA1537 at 5000 μg/plate in the presence of metabolite activation. Unexpected greater than 50% reduction of negative control revertants was observed in tester strain TA1537 at 50 μg/plate but would not consider cytotoxicity in the absence of metabolite activation. Results showed that CJ313 did not increase the number of revertants in all five tester strains TA98, TA100, TA102, TA1535 and TA1537 in the absence of metabolite activation, three tester strains TA98, TA100 and TA1535 in the presence of metabolite activation up to 5000 μg/plate; two tester strains TA102 and TA1537 in the presence of metabolite activation up to 1500 μg/plate. Based on the data obtained from this study, it was concluded that under the test condition, CJ313 was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 1500 μg/plate in the absence and presence of S9 metabolic activation.