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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

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(OECD TG471).

Everdirect SH12 was negative effect under the condition of in vitro mammalian chromosome aberration test (OECD TG473).

Everdirect SH12 was negative effect under the condition of in vitro mammalian cell gene mutation test (OECD TG476).

Link to relevant study records

Reference

Reference 1

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

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:

S9 Mix

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

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. 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).

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From December 02, 2015 to December 14, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

CHO-K1

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

CHO-K1

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Table 1. Cell viability test

Processing Time	Groups	Absorbance (570 nm)^a	Cell Viability (%)^b
Processed for 3 hours (with S9 Mix)	Negative control^c	0.928 ± 0.069	100.0
	Positive control^d	1.097 ± 0.022	118.2 ± 2.4
	Test group (mg/mL)
	2.0	0.457 ± 0.012	49.3 ± 1.3
	1.0	1.141 ± 0.006	122.9 ± 0.7
	0.5	1.034 ± 0.032	111.4 ± 3.5
	0.25	1.071 ± 0.113	115.4 ± 12.1
	0.125	1.233 ± 0.079	132.9 ± 8.5
Processed for 3 hours (without S9 Mix)	Negative control	1.878 ± 0.167	100.0
	Positive control	1.316 ± 0.038	70.0 ± 2.1
	Test group (mg/mL)
	2.0	0.571 ± 0.060	30.4 ± 3.2
	1.0	1.326 ± 0.041	70.6 ± 2.1
	0.5	1.270 ± 0.040	67.6 ± 2.1
	0.25	1.277 ± 0.143	68.0 ± 7.6
	0.125	1.352 ± 0.123	72.0 ± 6.5
Processed for 20 hours (without S9 Mix)	Negative control	1.556 ± 0.061	100.0
	Positive control	1.351 ± 0.155	86.8 ± 10.0
	Test group (mg/mL)
	2.0	0.266 ± 0.005	17.1 ± 0.3
	1.0	0.786 ± 0.065	50.5 ± 4.2
	0.5	0.922 ± 0.023	59.3 ± 1.4
	0.25	1.074 ± 0.096	69.0 ± 6.1
	0.125	1.207 ± 0.160	77.6 ± 10.3

^a Values were expressed as Mean ± S.D., and tests were triplicate.

^b Cell viability = each absorbance of the positive control or test groups / the average of absorbance of the negative control × 100%, than calculated the Mean ± S.D..

^c Negative control: Ham’s F-12 medium with 1% DMSO and 10% FBS (with or without S9 Mix).

^d Positive control: Group included 25μg/mL cyclophosphamide monohydrate for the cells treated with S9 Mix, and 2.5μg/mL mitomycin C for the cells treated without S9 Mix.

 

Table 2. The cell proportion with abnormal chromosome

Group	The cell proportion with abnormal chromosome (%)
	Processed for 3 hours (with S9 Mix)	Processed for 3 hours (without S9 Mix)	Processed for 20 hours (without S9 Mix)
Negative control	0.0	0.0	0.0
Positive control	10.3	8.0	18.0
Test group (mg/mL)
2.0	0.0	0.7	0.3
1.0	0.0	0.0	0.0
0.5	0.0	0.0	0.0

^a Tests were repeated triplicate.

^b Negative control: Ham’s F-12 medium with 1% DMSO and 10% FBS (with or without S9 Mix).

^c Positive control: Group included 25μg/mL cyclophosphamide monohydrate for the cells treated with S9 Mix, and 2.5μg/mL mitomycin C for the cells treated without S9 Mix.

Conclusions:

According to OECD 473 test method, Everdirect SH12 was negative effect under the condition of in vitro mammalian chromosome aberration test.

Executive summary:

This test using the procedures outlined in the SuperLub Study Plan for M62-151100085001EN which is based on the SOP for the OECD 473 (SOPF-207) and OECD 473 (OECD, 2014). The results of this OECD 473 test for Everdirect SH12 show that test validity criteria was met.

Based on the results of the cell viability test, 2.0mg/mL was set as the highest dose in this study.In the chromosome aberration test, three doses of Everdirect SH12 at 0.5, 1.0 and 2.0mg/mL, negative and positive controls were tested in triplicate with or without S9 Mix. The cell proportion with abnormal chromosome was lower than 3% for all test groups in the absence and presence of S9 Mix. Based on the data obtained from this study, it was concluded that under the test condition, Everdirect SH12 was negative effect in mammalian chromosome aberration test (in vitro).

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From July 04, 2016 to December 30, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

CHO-K1

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

CHO-K1

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Table 1. Cell viability analysis

Group	Test article	Average colony numbers^a	Relative survival (%)^b
With S9 Mix	Negative control^c	176.6 ± 2.7	100.0
	Positive control^d	166.6 ± 6.7	94.3 ± 3.8
	Test groups (mg/mL)
	2.0	198.0 ± 8.8	112.1 ± 5.0
	1.0	218.0 ± 6.5	123.4 ± 3.7
	0.5	199.2 ± 3.0	112.8 ± 1.7
	0.25	203.8 ± 4.1	115.4 ± 2.3
Without S9 Mix	Negative control^c	196.0 ± 5.3	100.0
	Positive control^d	140.2 ± 6.5	71.5 ± 3.3
	Test groups (mg/mL)
	2.0	187.8 ± 6.4	95.8 ± 3.3
	1.0	204.4 ± 4.0	104.3 ± 2.1
	0.5	183.6 ± 2.7	93.7 ± 1.4
	0.25	185.2 ± 1.6	94.5 ± 0.8

^a Values were expressed as Mean ± S.D., and tests were repeated 5 times.

^b Relative survival = each colony numbers of the positive control or test groups / the average of colony numbers in the negative control × 100%, then calculated the Mean ± S.D..

^c Negative control: Ham’s F-12 medium with 1% DMSO and 10% FBS (S9 Mix or not).

^d Positive control: 4μg/mL B[a]P for the cell treated with S9 Mix, and 0.25μg/mL 4-NQO for the cells treated without S9 Mix.

Table 2. Mutation frequency analysis

Group	Test article	Average colony numbers^a	Mutation frequency (× 10^-6)^b
With S9 Mix	Negative control^c	2.8 ± 2.3	5.0
	Positive control^d	91.6 ± 16.5	247.6*
	Test groups (mg/mL)
	2.0	3.2 ± 1.8	4.7
	1.0	4.0 ± 2.0	8.9
	0.5	3.2 ± 1.8	6.4
	0.25	4.4 ± 3.0	6.1
Without S9 Mix	Negative control^c	13.0 ± 1.4	29.0
	Positive control^d	42.6 ± 16.9	116.1*
	Test groups (mg/mL)
	2.0	19.6 ± 4.5	43.9
	1.0	16.6 ± 4.2	33.5
	0.5	10.2 ± 1.8	14.5
	0.25	10.0 ± 2.0	17.5

^a Values were expressed as Mean ± S.D., and tests were repeated 5 times.

^b Mutation frequency = (average colony numbers of each test groups / the number of seeding) × (1 / Colonies formation frequency).

^c Negative control: Ham’s F-12 medium with 1% DMSO and 10% FBS (S9 Mix or not).

^dPositive control: 4μg/mL B[a]P for the cell treated with S9 Mix, and 0.25μg/mL 4-NQO for the cells treated without S9 Mix.

* Significantly different from the negative control group (ρ < 0.005).

Conclusions:

According to OECD 476 test method, Everdirect SH12 was negative effect under the condition of in vitro mammalian cell gene mutation test.

Executive summary:

This test using the procedures outlined in the SuperLub Study Plan for M62-151100091001EN which is based on the SOP for the OECD 476 (SOPF-240) and OECD 476 (OECD, 2015). The results of this OECD 476 test for Everdirect SH12 show that test validity criteria was met.

Based on the results of the cell viability test, 2.0mg/mL was set as the highest dose in this study. In the gene mutation test, four doses of Everdirect SH12 at 0.25, 0.5, 1.0 and 2.0mg/mL, negative and positive controls were tested in five repetitions with or without S9 Mix. The mutation frequency was no significantly different from the negative control group for all test groups in the absence and presence of S9 Mix. Based on the data obtained from this study, it was concluded that under the test condition, Everdirect SH12 was negative effect in mammalian cell gene mutation test (in vitro).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Bacterial reverse mutation test (OECD TG471)

Based on the preliminary assay results, 5mg/plate was 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.

 

Mammalian chromosomal aberration test (OECD TG473)

Based on the results of the cell viability test, 2.0mg/mL was set as the highest dose in this study. In the chromosome aberration test, three doses of Everdirect SH12 at 0.5, 1.0 and 2.0mg/mL, negative and positive controls were tested in triplicate with or without S9 Mix. The cell proportion with abnormal chromosome was lower than 3% for all test groups in the absence and presence of S9 Mix. Based on the data obtained from this study, it was concluded that under the test condition, Everdirect SH12 was negative effect in vitro mammalian chromosome aberration test.

 

Mammalian cell gene mutation tests(OECD TG476)

Based on the results of the cell viability test, 2.0mg/mL was set as the highest dose in this study. In the gene mutation test, four doses of Everdirect SH12 at 0.25, 0.5, 1.0 and 2.0mg/mL, negative and positive controls were tested in five repetitions with or without S9 Mix. The mutation frequency was no significantly different from the negative control group for all test groups in the absence and presence of S9 Mix. Based on the data obtained from this study, it was concluded that under the test condition, Everdirect SH12 was negative effect in mammalian cell gene mutation test (in vitro).