7-({[3-({4-[(2-hydroxy-1-naphthyl)diazenyl]phenyl}diazenyl)phenyl]sulfonyl}amino)naphthalene-1,3-disulfonic acid, lithium sodium salts

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

CJ321 was considered a suspect or equivocal results in the reverse mutation analysis of Salmonella typhimurium (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 November 28, 2016 to June 02, 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:

The post-mitochondrial fraction (S9) prepared from uninduced male Golden Syrian Hamster liver

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

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

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	+	+	+	+	+
rfa mutation	inhibition zone of crystal violet	+	+	+	+	+
ΔuvrB 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 CJ321 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	35	29	89	79	74	410	345	358	18	16	17	15	8	10
	M^f	31 ± 3			81 ± 8			371 ± 34			17 ± 1			11 ± 4
50	I^e	28	23	38	79	63	82	348	345	327	14	16	19	15	17	18
	M^f	30 ± 8			75 ± 10			340 ± 11			16 ± 3			17 ± 2
150	I^e	24	37	26	63	68	66	354	305	325	21	17	24	18	19	12
	M^f	29 ± 7			66 ± 3			328 ± 25			21 ± 4			16 ± 4
500	I^e	24	36	32	43	59	54	347	356	309	16	10	20	6	16	11
	M^f	31 ± 6			52 ± 8			337 ± 25			15 ± 5			11 ± 5
1500	I^e	32	38	30	48	48	57	329	309	323	15	14	20	14	14	12
	M^f	33 ± 4			51 ± 5			320 ± 10			16 ± 3			13 ± 135
5000	I^e	35	32	28	56	70	38	269	274	283	16	16	16	5	11	13
	M^f	32 ± 4			55 ± 16			275 ± 7			16 ± 0			10 ± 4
Positive control^b	I^e	193	278	208	572	539	568	1966	1772	1896	472	489	419	172	168	151
	M^f	226^c± 45			560^c±18			1878^c± 98			460^d± 37			164^d± 11

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 CJ321 in Salmonella typhimurium Strains with S9 Metabolic Activation

Treatment (μg/plate)		Number of Revertant Colonies in Salmonella typhimurium
		TA98			TA100			TA102			TA1535			TA1537			TA98 (repeat)
Replicate		1	2	3	1	2	3	1	2	3	1	2	3	1	2	3	1	2	3
Negative control^a	I^e	61	63	52	105	107	89	393	382	422	14	10	4	19	20	26	46	41	43
	M^f	59 ± 6			100 ± 10			399 ± 21			9 ± 5			22 ± 4			43 ± 3
50	I^e	66	69	52	105	79	95	194	361	359	13	11	15	25	18	33	66	37	48
	M^f	62 ± 9			93 ± 13			305 ± 96			13 ± 2			25 ± 8			50 ± 15
150	I^e	73	99	134	102	99	101	386	371	238	19	13	12	20	28	29	107	77	145
	M^f	102 ± 31			101 ± 2			332 ± 81			15 ± 4			26 ± 5			110^c± 34
500	I^e	198	195	175	139	134	104	437	385	312	11	14	15	35	24	41	156	133	179
	M^f	189^c± 13			126 ± 19			378 ± 63			13 ± 2			33 ± 9			156^c± 23
1500	I^e	84	91	96	102	110	80	444	271	294	17	12	9	35	29	38	76	79	93
	M^f	90 ± 6			97 ± 16			336 ± 94			13 ± 4			34 ± 5			83 ± 9
5000	I^e	52	54	55	72	92	83	386	328	342	13	15	13	22	19	21	31	40	30
	M^f	54 ± 2			82 ± 10			352 ± 30			14 ± 1			21 ± 2			34 ± 6
Positive control^b	I^e	277	272	277	229	297	399	1093	1256	1402	306	303	392	386	303	244	233	278	212
	M^f	275^c± 3			308^c± 86			1250^c± 155			334^d± 51			311^d± 71			241^c± 34

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, CJ321 was considered a suspect or equivocal results in the reverse mutation analysis of Salmonella typhimurium.

Executive summary:

This test using the procedures outlined in the QPS Taiwan Study Plan for T65316021-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 CJ321 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 CJ321 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. Results showed that CJ321 did not increase the number of revertants in all five tester strains TA98, TA100, TA102, TA1535 and TA1537 up to 5000 μg/plate in the absence of metabolite activation. However,treatment with CJ321 in the presence of S9 metabolic activation increased the number of revertants in tester strain of Salmonella typhimurium TA98 at the dose 500μg/plate. The increase was not dose-responsiveness which led to an equivocal result. An equivocal result was also shown in the repeat (confirmation) study.

Based on the data obtained from these studies, it was concluded that under the test condition, CJ321 was considered a suspect or equivocal results in the reverse mutation analysis of Salmonella typhimurium.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

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 CJ321 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.Results showed that CJ321 did not increase the number of revertants in all five tester strains TA98, TA100, TA102, TA1535 and TA1537 up to 5000 μg/plate in the absence of metabolite activation. However,treatment with CJ321 in the presence of S9 metabolic activation increased the number of revertants in tester strain of Salmonella typhimurium TA98 at the dose 500 μg/plate. The increase was not dose-responsiveness which led to an equivocal result. An equivocal result was also shown in the repeat (confirmation) study.

Based on the data obtained from these studies, it was concluded that under the test condition, CJ321 was considered a suspect or equivocal results in the reverse mutation analysis of Salmonella typhimurium.