1-amino-4-({3-[(4-chloro-6-{[(3 or 4)-sulfophenyl]amino}-1,3,5-triazin-2-yl)amino]-2,4,6-trimethyl-5-sulfophenyl}amino)-9,10-dioxo-9,10-dihydroanthracene-2-sulfonic acid, lithium sodium salts

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

CJ304 was not mutagenic in the reverse mutation analysis of Salmonella typhimuriumup to 5000μg/plate in the absence and presence of S9 metabolic activation (OECD TG471).

The main structure of the read-across substance is the same of the CJ304, only the salt form is different. However, this difference does not influence the toxicity of the CJ304. CJ304 contains Li/Na salt form and the read-across substances contains only Na salt. This will not lead to difference on the toxicity results.

A second study was conducted according to OECD guideline no. 476 and in accordance with GLP. In a mammalian cell gene mutation assay (HPRT locus), CHO cells cultured in vitro were exposed to FAT 41001/H TE dissolved in culture medium at various concentrations. In the described in vitro cell gene mutagenicity test under the experimental conditions reported, the test item is considered to be non-mutagenic in the HPRT locus using CHO cells of the Chinese Hamster. A third study was conducted with the test item according to OECD guideline no. 473 and in accordance with GLP. The test article FAT 41001/H was assessed for its potential toinduce structural chromosomal aberrations in CHO cells of the Chinese hamster in vitro in two independent experiments. The substanceis considered to be non-mutagenic in thischromosome aberration test. Results of the three studies indicate that the test item is not classified as genotoxic.

Justification for selection of genetic toxicity endpoint

All three in vitro studies (OECD 471, OECD 473 and OECD 476) were taken into consideration.

Short description of key information:

The test substance showed negative results in the Salmonella typhimurium reverse mutation assay (OECD 471), in a mammalian cell gene mutation assay (OECD 476) and in chromosome aberrations test (OECD 473).

Endpoint Conclusion: No adverse effect observed (negative)

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 April 20, 2016 to August 29, 2016

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 Aroclor 1254-induced Sprague-Dawley rat

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

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

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

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

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

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

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 CJ304 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	28	27	26	76	72	79	318	321	301	16	15	15	10	8	10
	M^f	27 ± 1			76 ± 4			313 ± 11			15 ± 1			9 ± 1
50	I^e	30	36	28	72	71	70	363	234	218	17	9	10	10	13	7
	M^f	31 ± 4			71 ± 1			272 ± 80			12 ± 4			10 ± 3
150	I^e	15	29	22	66	56	54	267	221	348	14	12	16	5	8	6
	M^f	22 ± 7			59 ± 6			279 ± 64			14 ± 2			6 ± 2
500	I^e	31	33	23	80	55	76	316	302	285	16	15	22	11	11	10
	M^f	29 ± 5			70 ± 13			301 ± 16			18 ± 4			11 ± 1
1500	I^e	26	18	20	75	67	63	310	305	333	19	17	21	11	9	10
	M^f	21 ± 4			68 ± 6			316 ± 15			19 ± 2			10 ± 1
5000	I^e	24	21	24	45	58	51	314	238	254	11	13	14	10	12	4
	M^f	23 ± 2			51±7			269 ± 40			13 ± 2			9 ± 4
Positive control^b	I^e	152	154	170	403	401	538	732	834	824	440	406	417	87	90	63
	M^f	159^c± 10			447^c± 79			797^c± 56			421^d± 17			80^d± 15

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 CJ304 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	25	33	31	73	72	89	312	355	386	4	13	5	14	13	12
	M^f	30 ± 4			85 ± 11			351 ± 37			7 ± 5			13 ± 1
50	I^e	39	33	30	87	84	77	319	288	307	8	11	6	13	12	11
	M^f	34 ± 5			83 ± 5			305 ± 16			8 ± 3			12 ± 1
150	I^e	31	34	25	76	83	83	277	300	231	14	6	10	16	17	9
	M^f	30 ± 5			81 ± 4			269 ± 35			10 ± 4			14 ± 4
500	I^e	23	34	29	75	67	93	319	337	332	10	12	19	21	11	20
	M^f	29 ± 6			78 ± 13			329 ± 9			14 ± 5			17 ± 6
1500	I^e	39	24	22	81	63	59	335	286	336	9	12	11	16	11	10
	M^f	28 ± 9			68 ± 12			319 ± 29			11 ± 2			12 ± 3
5000	I^e	14	27	25	78	60	60	245	276	285	6	8	7	8	10	7
	M^f	22 ± 7			66 ± 10			269 ± 21			7 ± 1			8 ± 2
Positive control^b	I^e	210	207	184	667	676	698	1439	1685	1594	204	218	183	287	278	279
	M^f	200^c± 14			680^c± 16			1573^c± 124			202^d± 18			281^d± 5

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

Conclusions:

According to OECD 471 test method, CJ304 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 T65315027-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 CJ304 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 CJ304 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 CJ304 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, CJ304 was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 5000μg/plate in the absence and presence of S9 metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Genetic toxicity in vitro

Based on the preliminary assay results, 5000μg/plate was set as the highest dose in this study. In the mutagenicity assay, five doses of CJ304 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. The results of concurrent positive and negative controls and three non-cytotoxic dose levels obtained supported the validity of the assay.

No cytotoxicity was observed in all five tester strains up to 5000μg/plate in the absence and presence of metabolite activations. Results showed that CJ304 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, CJ304 was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 5000μg/plate in the absence and presence of S9 metabolic activation.