Pyridinium, 1,1'-[(6,13-dichloro-4,11-disulfo-3,10-triphenodioxazinediyl)bis[imino-2,1-ethanediylimino[6-[(2,5-disulfophenyl)amino]-1,3,5-triazine-4,2-diyl]]]bis[3-carboxy-, dihydroxide, bis(inner salt), hexasodium salt

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Everzol SB26 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).

Everzol SB26 was negative effect under the condition of in vitro mammalian chromosome aberration test (OECD TG473).

Everzol SB26was 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 22, 2016 to December 09, 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	1234
	Test group
	5	177
	2.5	164
	1.25	212
	0.625	191
	0.3125	205
Without S9 Mix	Negative control group	171
	Positive control group	770
	Test group
	5	142
	2.5	137
	1.25	131
	0.625	138
	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	53	29	38	40 ± 12
	Positive control group^b	234	226	218	226 ± 8*
	Test group
	5	31	28	35	31 ± 4
	2.5	28	30	34	31 ± 3
	1.25	35	31	31	32 ± 2
	0.625	40	35	41	39 ± 3
	0.3125	57	46	42	48 ± 8
Without S9 Mix	Negative control group	31	40	23	31 ± 9
	Positive control group	283	257	228	256 ± 28*
	Test group
	5	27	25	28	27 ± 2
	2.5	34	22	35	30 ± 7
	1.25	31	27	24	27 ± 4
	0.625	31	29	35	32 ± 3
	0.3125	27	36	29	31 ± 5

^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	210	193	200	201 ± 9
	Positive control group^b	1332	1415	1372	1373 ± 42*
	Test group
	5	176	171	178	175 ± 4
	2.5	204	222	178	201 ± 22
	1.25	206	199	189	198 ± 9
	0.625	211	203	186	200 ± 13
	0.3125	219	204	192	205 ± 14
Without S9 Mix	Negative control group	214	204	185	201 ± 15
	Positive control group	842	819	827	829 ± 12*
	Test group
	5	209	235	195	213 ± 20
	2.5	205	195	209	203 ± 7
	1.25	187	223	212	207 ± 18
	0.625	209	204	219	211 ± 8
	0.3125	216	177	233	209 ± 29

^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	486	435	461	461 ± 26
	Positive control group^b	1065	1136	1073	1091 ± 39*
	Test group
	5	387	409	393	396 ± 11
	2.5	442	454	470	455 ± 14
	1.25	407	383	428	406 ± 23
	0.625	426	409	461	432 ± 27
	0.3125	489	466	446	467 ± 22
Without S9 Mix	Negative control group	400	339	372	370 ± 31
	Positive control group	839	834	870	848 ± 20*
	Test group
	5	371	363	357	364 ± 7
	2.5	353	375	336	355 ± 20
	1.25	372	390	361	374 ± 15
	0.625	418	357	404	393 ± 32
	0.3125	390	315	375	360 ± 40

^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	20	15	24	20 ± 5
	Positive control group^b	278	301	285	288 ± 12*
	Test group
	5	23	17	16	19 ± 4
	2.5	14	25	18	19 ± 6
	1.25	27	20	22	23 ± 4
	0.625	16	17	29	21 ± 7
	0.3125	29	27	23	26 ± 3
Without S9 Mix	Negative control group	22	21	20	21 ± 1
	Positive control group	259	244	246	250 ± 8*
	Test group
	5	18	26	22	22 ± 4
	2.5	20	24	25	23 ± 3
	1.25	28	30	19	26 ± 6
	0.625	25	18	31	25 ± 7
	0.3125	30	27	25	27 ± 3

^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	20	18	17	18 ± 2
	Positive control group^b	477	445	451	458 ± 17*
	Test group
	5	10	7	13	10 ± 3
	2.5	12	17	11	13 ± 3
	1.25	15	22	23	20 ± 4
	0.625	16	15	10	14 ± 3
	0.3125	11	13	19	14 ± 4
Without S9 Mix	Negative control group	18	16	16	17 ± 1
	Positive control group	606	768	713	696 ± 82*
	Test group
	5	13	10	20	14 ± 5
	2.5	17	11	19	16 ± 4
	1.25	10	9	11	10 ± 1
	0.625	14	13	12	13 ± 1
	0.3125	18	15	23	19 ± 4

^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, Everzol SB26 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 M62-151100132001EN 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 Everzol SB26 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 Everzol SB26 at 0.3125, 0.625, 1.25, 2.5 and 5 mg/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.

In the range-finding test of the Everzol SB26, no toxicity was observed in all five tester strains up to 5mg/plate in the absence and presence of metabolite activations. Results showed that Everzol SB26 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, Everzol SB26 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:

mitomycin C

other: cyclophosphamide monohydrate

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

CHO-K1

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. Cell viability test

Processing Time	Groups	Absorbance (570 nm)^a	Cell Viability (%)^b
Processed for 3 hours (with S9 Mix)	Negative control^c	0.997 ± 0.089	100.0
	Positive control^d	0.906±0.094	90.8 ± 9.4
	Test group (mg/mL)
	2.0	0.761 ± 0.111	76.3 ± 11.2
	1.0	0.839 ± 0.066	84.1 ± 6.7
	0.5	0.647 ± 0.055	64.9 ± 5.5
	0.25	0.798 ± 0.078	80.0 ± 7.8
	0.125	1.268 ± 0.108	127.2 ± 10.8
Processed for 3 hours (without S9 Mix)	Negative control	1.694 ± 0.202	100.0
	Positive control	1.354 ± 0.180	79.9 ± 10.6
	Test group (mg/mL)
	2.0	1.278 ± 0.070	75.5 ± 4.2
	1.0	1.494 ± 0.152	88.2 ± 8.9
	0.5	1.284 ± 0.062	75.8 ± 3.7
	0.25	1.233 ± 0.014	72.8 ± 0.8
	0.125	1.247 ± 0.181	73.6 ± 10.7
Processed for 20 hours (without S9 Mix)	Negative control	1.686 ± 0.169	100.0
	Positive control	1.563 ± 0.035	92.7 ± 2.1
	Test group (mg/mL)
	2.0	1.686 ± 0.037	100.0 ± 2.2
	1.0	1.523 ± 0.110	90.3 ± 6.5
	0.5	1.327 ± 0.114	78.7 ± 6.8
	0.25	1.300 ± 0.068	77.1 ± 4.1
	0.125	1.106 ± 0.066	65.6 ± 3.9

^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	8.7	8.0	13.7
Test group (mg/mL)
2.0	0.0	0.0	0.7
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, Everzol SB26 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 M62-151100133001EN 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 Everzol SB26 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 Everzol SB26 at 0.5, 1.0 and 2.0 mg/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, Everzol SB26 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 19, 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 ± 3.6	100.0
	Positive control^d	165.4 ± 6.7	93.7 ± 3.8
	Test groups (mg/mL)
	2.0	199.8 ± 11.4	113.1 ± 6.4
	1.0	214.8 ± 7.9	121.6 ± 4.4
	0.5	196.8 ± 4.4	111.4 ± 2.5
	0.25	203.8 ± 6.1	115.4 ± 3.5
Without S9 Mix	Negative control^c	192.6 ± 5.5	100.0
	Positive control^d	140.0 ± 9.8	72.7 ± 5.1
	Test groups (mg/mL)
	2.0	187.6 ± 9.1	97.4 ± 4.7
	1.0	202.2 ± 5.6	105.0 ± 2.9
	0.5	183.6 ± 2.7	95.3 ± 1.4
	0.25	185.6 ± 2.6	96.4 ± 1.4

^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	6.4 ± 1.3	17.6
	Positive control^d	35.2 ± 4.8	110.7*
	Test groups (mg/mL)
	2.0	5.8 ± 2.4	18.0
	1.0	4.0 ± 2.0	8.7
	0.5	4.4 ± 1.9	7.0
	0.25	2.8 ± 1.3	5.4
Without S9 Mix	Negative control^c	7.2 ± 0.8	13.2
	Positive control^d	41.8 ± 6.1	92.3*
	Test groups (mg/mL)
	2.0	10.6 ± 1.5	18.0
	1.0	11.4 ± 3.6	16.1
	0.5	8.8 ± 1.3	13.6
	0.25	4.2 ± 0.8	6.6

^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).

^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.

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

Conclusions:

According to OECD 476 test method, Everzol SB26 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 M62-151100141001EN 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 Everzol SB26 show that test validity criteria was met.

Based on the results of the cell viability test, 2.0 mg/mL was set as the highest dose in this study. In the gene mutation test, four doses of Everzol SB26 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, Everzol SB26 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 Everzol SB26 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. In the range-finding test for toxicity of the Everzol SB26, no toxicity was observed in all five tester strains up to 5 mg/plate in the absence and presence of metabolite activations. Results showed that Everzol SB26 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, Everzol SB26 was not mutagenic in the reverse mutation analysis of Salmonella typhimuriumup 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, 2.0 mg/mL was set as the highest dose in this study. In the chromosome aberration test, three doses of Everzol SB26 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, Everzol SB26 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, 2.0 mg/mL was set as the highest dose in this study. In the gene mutation test, four doses of Everzol SB26 at 0.25, 0.5, 1.0 and 2.0 mg/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, Everzol SB26 was negative effect in mammalian cell gene mutation test (in vitro).