CJ309

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 February 18, 2016 to August 29, 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:

Reaction products of 2,7-Naphthalenedisulfonic acid, 4-amino-6-[2-[5-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-2-sulfophenyl]diazenyl]-3-[2-(2,5-disulfophenyl)diazenyl]-5-hydroxy-, pentasodium salt and lithium chloride

Molecular formula:

Not applicable; this UVCB substance contains: C25H14ClN10O16S5.xLi.yNa, (x + y) = 5; 0 < (x,y) < 5 with 940.9 < MW < 1021.1 g/mol (UVCB substance), and traces of NaCl.

IUPAC Name:

Reaction products of 2,7-Naphthalenedisulfonic acid, 4-amino-6-[2-[5-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-2-sulfophenyl]diazenyl]-3-[2-(2,5-disulfophenyl)diazenyl]-5-hydroxy-, pentasodium salt and lithium chloride

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Method

Target gene:

Bacterial gene reverse mutation

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 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	+	+	+	+	+
△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 CJ309 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	18	23	25	82	79	60	380	407	433	16	13	23	9	25	8
	M^f	22 ± 4			74 ± 12			407 ± 27			17 ± 5			14 ± 10
50	I^e	35	25	17	68	81	71	402	314	345	20	10	16	9	16	14
	M^f	26 ± 9			73 ± 7			354 ± 45			15 ± 5			13 ± 4
150	I^e	25	14	25	70	71	67	335	343	336	14	24	12	15	16	22
	M^f	21 ± 6			69 ± 2			338 ± 4			17 ± 6			18 ± 4
500	I^e	26	23	22	86	66	66	364	315	317	24	25	23	13	13	16
	M^f	24 ± 2			73 ± 12			332 ± 28			24 ± 1			14 ± 2
1500	I^e	29	24	24	71	67	70	399	324	364	22	16	14	19	17	8
	M^f	26 ± 3			69 ± 2			362 ± 38			17 ± 4			15 ± 6
5000	I^e	17	24	16	58	65	67	384	282	326	14	15	11	19	14	16
	M^f	19 ± 4			63±5			331 ± 51			13 ± 2			16 ± 3
Positive control^b	I^e	224	217	196	575	587	591	1218	1018	1182	380	355	416	171	143	140
	M^f	212^c± 15			584^c± 8			1139^c± 107			384^d± 31			151^d± 17

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 CJ309 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	46	49	44	73	87	78	344	427	377	10	8	10	13	10	13
	M^f	46 ± 3			79 ± 7			383 ± 42			9 ± 1			12 ± 2
50	I^e	39	40	51	77	70	67	342	298	284	9	8	8	11	22	18
	M^f	43 ± 7			71 ± 5			308 ± 30			8 ± 1			17 ± 6
150	I^e	49	45	41	86	86	75	281	310	232	6	7	9	9	16	10
	M^f	45 ± 4			82 ± 6			274 ± 39			7 ± 2			12 ± 4
500	I^e	44	36	38	76	70	76	266	343	316	5	5	8	6	11	12
	M^f	39 ± 4			74 ± 3			308 ± 39			6 ± 2			10 ± 3
1500	I^e	48	37	35	80	65	64	324	454	364	8	12	10	13	8	19
	M^f	40 ± 7			70 ± 9			381 ± 67			10 ± 2			13 ± 6
5000	I^e	23	30	45	64	70	64	263	431	382	7	9	1	7	9	10
	M^f	33 ± 11			66 ± 3			359 ± 86			6 ± 4			9 ± 2
Positive control^b	I^e	248	229	225	610	651	642	1424	1350	1688	272	287	249	270	316	307
	M^f	234^c± 12			634^c± 22			1487^c± 178			269^d± 19			298^d± 24

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, CJ309 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 T65315029-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 CJ309 show that test validity criteria was met.

Based on the preliminary assay results, 5000μg/plate was set as the highest dose in this study. In the mutagenicity assay, five doses of CJ309 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 CJ309 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, CJ309 was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 5000μg/plate in the absence and presence of S9 metabolic activation.