Cuprate(4-), [μ-[[7,7'-[[6-[(2-hydroxyethyl)amino]-1,3,5-triazine-2,4-diyl]diimino]bis[4-(hydroxy-κO)-3-[[2-(hydroxy-κO)-5-sulfophenyl]azo-κN1]-2-naphthalenesulfonato]](8-)]]di-, tetrasodium

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 August 8, 2016 to November 18, 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:

Cuprate(4-), [μ-[[7,7'-[[6-[(2-hydroxyethyl)amino]-1,3,5-triazine-2,4-diyl]diimino]bis[4-(hydroxy-κO)-3-[[2-(hydroxy-κO)-5-sulfophenyl]azo-κN1]-2-naphthalenesulfonato]](8-)]]di-, tetrasodium

EC Number:

820-015-3

Cas Number:

129874-15-3

Molecular formula:

C37H22Cu2N10O17S4.4Na

IUPAC Name:

Cuprate(4-), [μ-[[7,7'-[[6-[(2-hydroxyethyl)amino]-1,3,5-triazine-2,4-diyl]diimino]bis[4-(hydroxy-κO)-3-[[2-(hydroxy-κO)-5-sulfophenyl]azo-κN1]-2-naphthalenesulfonato]](8-)]]di-, tetrasodium

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Everdirect SH12
- Substance type: Powder
- Composition of test material, percentage of components: 69.96 %
- Lot/batch No.: 3506
- Storage condition of test material: Ambient

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:

S9 Mix

Controls

Untreated negative controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-Aminoanthracene

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. Characteristics of five Salmonella typhimurium strains

Test strain	Histidine requirement	uvrB mutation	rfa mutation	Ampicillin resistance
TA98	+	+	+	+
TA100	+	+	+	+
TA102	+	ϴ	+	+
TA1535	+	+	+	ϴ
TA1537	+	+	+	ϴ

+ means had the characteristic; ϴ means did not have the characteristic

Table 2. Toxicity of the test article of Salmonella typhimurium TA100

Group	Test article (mg/plate)	Reverse mutant colony number (CFU/plate)
With S9 Mix	Negative control group	200
	Positive control group^a	1292
	Test group
	5	194
	2.5	242
	1.25	225
	0.625	221
	0.3125	221
Without S9 Mix	Negative control group	218
	Positive control group	955
	Test group
	5	188
	2.5	193
	1.25	197
	0.625	172
	0.3125	171

^a Positive control group: With S9 Mix: 2-Aminoanthracene (4.0μg/plate).

Without S9 Mix: Sodium azide (5μg/plate).

Table 3. Reverse mutation test of Salmonella typhimurium TA98

Group	Test article (mg/plate)	Reverse mutant colony number (CFU/plate)			Average of colonies^a
		1	2	3
With S9 Mix	Negative control group	46	31	36	38 ± 8
	Positive control group^b	262	274	323	286 ± 32*
	Test group
	5	29	31	25	28 ± 3
	2.5	31	34	25	30 ± 5
	1.25	32	26	29	29 ± 3
	0.625	29	31	23	28 ± 4
	0.3125	29	23	24	29 ± 6
Without S9 Mix	Negative control group	53	41	42	45 ± 7
	Positive control group	298	317	291	302 ± 13*
	Test group
	5	45	43	48	45 ± 3
	2.5	59	40	37	45 ± 12
	1.25	52	50	43	48 ± 5
	0.625	43	33	37	38 ± 5
	0.3125	53	35	51	46 ± 10

^a Average of colonies was shown as Mean ± S.D., the data were triplicate.

^b Positive control group: With S9 Mix: Benzo [a] pyrene (4.0μg/plate).

Without S9 Mix: 4-Nitroquinoline-N-oxide (0.5μg/plate).

^*Reverse mutant colony number were twice more than negative control group, ρ < 0.05 (One-Way ANOVA).

Table 4. Reverse mutation test of Salmonella typhimurium TA100

Group	Test article (mg/plate)	Reverse mutant colony number (CFU/plate)			Average of colonies^a
		1	2	3
With S9 Mix	Negative control group	183	251	237	224 ± 36
	Positive control group^b	1317	1301	1270	1296 ± 24*
	Test group
	5	213	191	231	212 ± 20
	2.5	230	185	243	219 ± 30
	1.25	192	191	248	210 ± 33
	0.625	228	204	201	211 ± 15
	0.3125	221	237	189	216 ± 24
Without S9 Mix	Negative control group	253	223	211	229 ± 22
	Positive control group	947	969	891	936 ± 40*
	Test group
	5	209	200	196	202 ± 7
	2.5	184	244	198	209 ± 31
	1.25	240	197	190	209 ± 27
	0.625	207	188	187	194 ± 11
	0.3125	208	212	238	219 ± 16

^a Average of colonies was shown as Mean ± S.D., the data were triplicate.

^b Positive control group: With S9 Mix: 2-Aminoanthracene (4.0μg/plate).

Without S9 Mix: Sodium azide (5μg/plate).

^*Reverse mutant colony number were twice more than negative control group, ρ < 0.05 (One-Way ANOVA).

 

Table 5. Reverse mutation test of Salmonella typhimurium TA102

Group	Test article (mg/plate)	Reverse mutant colony number (CFU/plate)			Average of colonies^a
		1	2	3
With S9 Mix	Negative control group	468	395	482	448 ± 47
	Positive control group^b	1010	968	997	992 ± 22*
	Test group
	5	392	408	451	417 ± 31
	2.5	414	433	479	442 ± 33
	1.25	443	417	444	435 ± 15
	0.625	436	479	459	458 ± 22
	0.3125	515	465	439	473 ± 39
Without S9 Mix	Negative control group	375	359	405	380 ± 23
	Positive control group	976	906	945	942 ± 35*
	Test group
	5	482	464	494	480 ± 15
	2.5	469	444	490	468 ± 23
	1.25	391	489	445	442 ± 49
	0.625	412	442	500	451 ± 45
	0.3125	528	492	496	505 ± 20

^a Average of colonies was shown as Mean ± S.D., the data were triplicate.

^b Positive control group: With S9 Mix: 2-Aminoanthracene (10μg/plate).

Without S9 Mix: Mitomycin C (0.5μg/plate).

^*Reverse mutant colony number were twice more than negative control group, ρ < 0.05 (One-Way ANOVA).

 

Table 6. Reverse mutation test of Salmonella typhimurium TA1535

Group	Test article (mg/plate)	Reverse mutant colony number (CFU/plate)			Average of colonies^a
		1	2	3
With S9 Mix	Negative control group	22	30	19	24 ± 6
	Positive control group^b	229	264	243	245 ± 18*
	Test group
	5	26	28	25	26 ± 2
	2.5	30	24	33	29 ± 5
	1.25	31	26	21	26 ± 5
	0.625	19	23	32	25 ± 7
	0.3125	20	30	22	24 ± 5
Without S9 Mix	Negative control group	19	29	31	26 ± 6
	Positive control group	261	253	266	260 ± 7*
	Test group
	5	26	30	26	27 ± 2
	2.5	34	21	26	27 ± 7
	1.25	29	21	30	27 ± 5
	0.625	26	31	27	28 ± 3
	0.3125	29	30	19	26 ± 6

^a Average of colonies was shown as Mean ± S.D., the data were triplicate.

^b Positive control group: With S9 Mix: 2-Aminoanthracene (4.0μg/plate).

Without S9 Mix: Sodium azide (0.4μg/plate).

^*Reverse mutant colony number were twice more than negative control group, ρ < 0.05 (One-Way ANOVA).

 

Table 7. Reverse mutation test of Salmonella typhimurium TA1537

Group	Test article (mg/plate)	Reverse mutant colony number (CFU/plate)			Average of colonies^a
		1	2	3
With S9 Mix	Negative control group	10	17	16	14 ± 4
	Positive control group^b	289	333	314	312 ± 22*
	Test group
	5	9	15	11	12 ± 3
	2.5	9	12	15	12 ± 3
	1.25	11	16	14	14 ± 3
	0.625	21	21	15	19 ± 3
	0.3125	13	17	10	13 ± 4
Without S9 Mix	Negative control group	12	14	14	13 ± 1
	Positive control group	740	529	688	652 ± 110*
	Test group
	5	14	21	16	17 ± 4
	2.5	14	21	11	15 ± 5
	1.25	21	16	11	16 ± 5
	0.625	12	15	13	13 ± 2
	0.3125	11	23	17	17 ± 6

^a Average of colonies was shown as Mean ± S.D., the data were triplicate.

^b Positive control group: With S9 Mix: 2-Aminoanthracene (4.0μg/plate).

Without S9 Mix: 9-Aminoacridine (50.0μg/plate).

^*Reverse mutant colony number were twice more than negative control group, ρ < 0.05 (One-Way ANOVA).

Applicant's summary and conclusion

Conclusions:

According to OECD 471 test method, Everdirect SH12 was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 5 mg/plate.

Executive summary:

This test using the procedures outlined in the SuperLub Study Plan for M62-151100084001EN which is based on the SOP for the OECD 471 (SOPF-203) and OECD 471 (OECD, 1997). The results of this OECD 471 test for Everdirect SH12 show that test validity criteria was met.

Based on the preliminary assay results, 5mg/platewas set as the highest dose in this study. In the mutagenicity assay, five doses of Everdirect SH12 at 0.3125, 0.625, 1.25, 2.5 and 5mg/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 genetoxicity was observed in all five tester strains up to 5mg/plate in the absence and presence of metabolite activations. Results showed that Everdirect SH12 did not increase the number of revertants in all five tester strains TA98, TA100, TA102, TA1535 and TA1537 up to 5mg/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, Everdirect SH12 was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 5mg/plate in the absence and presence of S9 metabolic activation.