Sodium [4-[[dihydroxy[(2-hydroxy-3,5-dinitrophenyl)azo]phenyl]azo]benzenesulphonato(3-)]cuprate(1-)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Everlan SL65 was mutagenic in the reverse mutation analysis of Salmonella typhimurium (OECD TG471).

Everlan SL65 was positive effect under the condition of in vitro mammalian chromosome aberration test (OECD TG473).

Everlan SL65 was positive 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:

Sep. 20, 2016 - Dec 8, 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)

Version / remarks:

OECD 471:1997

Deviations:

no

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

Details on mammalian cell type (if applicable):

Source of Cell: MOLTOX, molecular Toxicology, Inc.

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-Aminoanthracene

Evaluation criteria:

for the groups treated with an without S9 mix, the range of natural reverse mutant colony number were as following: (CFU/plate)
Tester Strains With S9 mix without S9 mix
TA98 32-51 22-51
TA100 197-386 166-326
TA102 378-487 262-407
TA1535 18-41 17-43
TA1537 9-26 6-25

Statistics:

statistic via SPSS software, p < 0.05 (One-Way ANOVA).

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not determined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not determined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not determined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not determined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

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. 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	213
	Positive control group^a	1228
	Test group
	5	348
	2.5	716
	1.25	415
	0.625	353
	0.3125	282
Without S9 Mix	Negative control group	202
	Positive control group	781
	Test group
	5	486
	2.5	446
	1.25	350
	0.625	302
	0.3125	228

^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	43	32	51	42 ± 10
	Positive control group^b	353	378	347	359 ± 16*
	Test group
	5	102	109	92	101 ± 9*
	2.5	96	98	85	93 ± 7*
	1.25	101	87	130	106 ± 22*
	0.625	87	94	100	94 ± 7*
	0.3125	49	56	71	59 ± 11
Without S9 Mix	Negative control group	22	27	28	26 ± 3
	Positive control group	373	345	400	373 ± 28*
	Test group
	5	37	39	63	46 ± 14
	2.5	90	76	86	84 ± 7*
	1.25	59	67	76	67 ± 9*
	0.625	54	56	59	56 ± 3*
	0.3125	46	54	64	55 ± 9*

^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	171	191	235	199 ± 33
	Positive control group^b	1553	1647	1498	1566 ± 75*
	Test group
	5	485	362	414	420 ± 62*
	2.5	765	667	765	732 ± 57*
	1.25	691	619	561	624 ± 65*
	0.625	413	425	461	433 ± 25*
	0.3125	329	378	381	363 ± 29*
Without S9 Mix	Negative control group	201	147	164	171 ± 28
	Positive control group	738	797	759	765 ± 30*
	Test group
	5	498	422	560	493 ± 69*
	2.5	449	410	478	446 ± 34*
	1.25	488	479	555	507 ± 42*
	0.625	459	408	450	439 ± 27*
	0.3125	366	379	358	368 ± 11*

^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	482	478	410	457 ± 40
	Positive control group^b	1025	939	996	987 ± 44*
	Test group
	5	528	596	487	537 ± 55
	2.5	636	596	653	628 ± 29
	1.25	583	646	576	602 ± 39
	0.625	484	480	540	501 ± 34
	0.3125	621	530	491	547 ± 67
Without S9 Mix	Negative control group	401	365	446	404 ± 41
	Positive control group	925	920	844	896 ± 45*
	Test group
	5	533	534	383	483 ± 87
	2.5	528	462	505	498 ± 34
	1.25	589	461	490	513 ± 67
	0.625	485	515	467	489 ± 24
	0.3125	486	594	499	526 ± 59

^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	17	25	26	23 ± 5
	Positive control group^b	347	388	379	371 ± 22*
	Test group
	5	111	120	118	116 ± 5*
	2.5	164	139	131	145 ± 17*
	1.25	76	74	81	77 ± 4*
	0.625	59	54	74	62 ± 10*
	0.3125	37	45	39	40 ± 4
Without S9 Mix	Negative control group	22	23	24	23 ± 1
	Positive control group	228	235	213	225 ± 11*
	Test group
	5	40	38	44	41 ± 3
	2.5	113	138	92	114 ± 23*
	1.25	88	83	87	86 ± 3*
	0.625	54	53	55	54 ± 1*
	0.3125	49	42	39	43 ± 5

^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	14	9	12	12 ± 3
	Positive control group^b	450	476	438	455 ± 19*
	Test group
	5	89	96	74	86 ± 11*
	2.5	95	125	115	112 ± 15*
	1.25	63	94	102	86 ± 21*
	0.625	55	57	53	55 ± 2*
	0.3125	33	30	33	32 ± 2
Without S9 Mix	Negative control group	10	15	12	12 ± 3
	Positive control group	899	863	1020	927 ± 82*
	Test group
	5	78	85	66	76 ± 10*
	2.5	40	64	62	55 ± 13*
	1.25	59	86	37	61 ± 25*
	0.625	72	60	54	62 ± 9*
	0.3125	44	40	41	42 ± 2*

^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, Everlan SL65 was mutagenic in the reverse mutation analysis of Salmonella typhimurium.

Executive summary:

This test using the procedures outlined in the SuperLub Study Plan for M62-151100105001EN which is based on the SOP for the OECD 471 (SOPF-203) and OECD 471 (OECD, 1997). The results of this OECD 471 test forEverlan SL65show that test validity criteria was met.

Based on the preliminary assay results, 5mg/plate was set as the highest dose in this study. In the mutagenicity assay, five doses of Everlan SL65 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. At least one dose in four tester strains of Salmonella typhimurium TA98, TA100, TA1535 and TA1537 of test groups, the results showed that the number of revertants increase twice more than negative controls group. Based on the data obtained from this study, it was concluded that under the test condition, Everlan SL65 was mutagenic in the reverse mutation analysis of Salmonella typhimurium.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 25, 2016 - Dec 30, 2016

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OECD 476:2015

Deviations:

no

GLP compliance:

yes

Type of assay:

other: in vitro gene mutation study in mammalian cells

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

CHO-K1

Details on mammalian cell type (if applicable):

- Source of cells: Food Industry Research and Development Institute

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

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

CHO-K1

Metabolic activation:

with and without

Genotoxicity:

positive

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	167.4 ± 2.1	100.0
	Positive control^d	153.2 ± 4.7	91.5 ± 2.8
	Test groups (mg/mL)
	2.0	76.6 ± 3.8	45.8 ± 2.3
	1.0	184.2 ± 2.9	110.0 ± 1.7
	0.5	187.4 ± 5.8	111.9 ± 3.4
	0.25	184.4 ± 4.6	110.2 ± 2.7
Without S9 Mix	Negative control^c	195.8 ± 2.9	100.0
	Positive control^d	111.8 ± 11.3	57.1 ± 5.8
	Test groups (mg/mL)
	2.0	105.0 ± 6.0	53.6 ± 3.1
	1.0	158.8 ± 6.8	81.1 ± 3.5
	0.5	154.0 ± 1.6	78.7 ± 0.8
	0.25	183.4 ± 7.1	93.7 ± 3.6

^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	7.4 ± 0.9	18.9
	Positive control^d	61.0 ± 9.9	187.7*
	Test groups (mg/mL)
	2.0	45.0 ± 11.0	98.3*
	1.0	25.4 ± 4.7	49.9*
	0.5	13.2 ± 3.1	58.4*
	0.25	15.6 ± 1.7	27.3
Without S9 Mix	Negative control^c	9.6 ± 2.3	16.0
	Positive control^d	142.0 ± 20.3	272.0*
	Test groups (mg/mL)
	2.0	18.6 ± 3.9	35.0
	1.0	13.6 ± 3.8	20.7
	0.5	11.0 ± 2.0	26.7
	0.25	12.8 ± 3.3	27.0

^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, Everlan SL65 was positive 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 M62-151100112001EN 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 Everlan SL65 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 Everlan SL65 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 significantly different from the negative control group for 0.5-2.0 mg/mL of the test groups in the presence of S9 Mix. Based on the data obtained from this study, it was concluded that under the test condition, Everlan SL65 was positive effect in mammalian cell gene mutation test (in vitro).

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Dec 02,2015 - Dec 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)

Version / remarks:

OECD 473:2014

Deviations:

no

GLP compliance:

yes

Type of assay:

other: In vitro Mammalian Chromosome Aberration Test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

CHO-K1

Details on mammalian cell type (if applicable):

- Source of cells: Food Industry Research and Development Institute

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

CHO-K1

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

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	1.009 ± 0.135	100.0
	Positive control^d	1.038 ± 0.080	102.8 ± 7.9
	Test group (mg/mL)
	2.0	0.479 ± 0.010	47.5 ± 1.0
	1.0	1.199 ± 0.020	118.8 ± 2.0
	0.5	1.045 ± 0.024	103.6 ± 2.4
	0.25	1.103 ± 0.114	109.3 ± 11.2
	0.125	1.236 ± 0.063	122.5 ± 6.2
Processed for 3 hours (without S9 Mix)	Negative control	1.933 ± 0.183	100.0
	Positive control	1.209 ± 0.218	62.5 ± 11.2
	Test group (mg/mL)
	2.0	0.553 ± 0.065	28.6 ± 3.4
	1.0	1.201 ± 0.197	62.1 ± 10.2
	0.5	1.175 ± 0.215	60.8 ± 11.1
	0.25	1.288 ± 0.162	66.6 ± 8.4
	0.125	1.309 ± 0.242	67.7 ± 12.5
Processed for 20 hours (without S9 Mix)	Negative control	1.670 ± 0.130	100.0
	Positive control	1.192 ± 0.105	71.4 ± 6.3
	Test group (mg/mL)
	2.0	0.092 ± 0.006	5.5 ± 0.4
	1.0	0.329 ± 0.037	19.7 ± 2.2
	0.5	1.168 ± 0.215	69.9 ± 12.9
	0.25	1.102 ± 0.100	66.0 ± 5.9
	0.125	1.241 ± 0.063	74.3 ± 3.8

^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	13.3	10.3	19.3
Test group (mg/mL)
2.0	4.0	1.3	─
1.0	7.0	2.0	7.3
0.5	4.0	0.3	1.3
0.25	─	─	0.7

^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, Everlan SL65 was positive 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 M62-151100106002EN 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 Everlan SL65 show that test validity criteria was met.

Based on the results of the cell viability test, 2.0 mg/mL as the highest dosage was used to perform the test in 3-hours treatment with or without S9 Mix and 1.0 mg/mL as the highest dosage was used to perform the test in 20-hours treatment without S9 Mix. In the chromosome aberration test, three doses of Everlan SL65, negative and positive controls were tested in triplicate with or without S9 Mix. In test group, the cell proportions with abnormal chromosome in all dosage with S9 Mix for 3 hours and in highest dosage without S9 Mix for 20 hours gave the similar reaction as the positive controls. Based on the data obtained from this study, it was concluded that under the test condition, Everlan SL65 was positive effect in mammalian chromosome aberration test (in vitro).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available (further information necessary)

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 Everlan SL65 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. At least one dose in four tester strains of Salmonella typhimurium TA98, TA100, TA1535 and TA1537 of test groups, the results showed that the number of revertants increase twice more than negative controls group. Based on the data obtained from this study, it was concluded that under the test condition, Everlan SL65 was mutagenic in the reverse mutation analysis of Salmonella typhimurium.

 

Mammalian chromosomal aberration test (OECD TG473)

Based on the results of the cell viability test, 2.0 mg/mL as the highest dosage was used to perform the test in 3-hours treatment with or without S9 Mix and 1.0 mg/mL as the highest dosage was used to perform the test in 20-hours treatment without S9 Mix.In the chromosome aberration test, three doses of Everlan SL65, negative and positive controls were tested in triplicate with or without S9 Mix. In test group, the cell proportions with abnormal chromosome in all dosage with S9 Mix for 3 hours and in highest dosage without S9 Mix for 20 hours gave the similar reaction as the positive controls.

Based on the data obtained from this study, it was concluded that under the test condition, Everlan SL65 was positive effect in mammalian chromosome aberration test (in vitro).

 

Mammalian cell gene mutation test (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 Everlan SL65 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 significantly different from the negative control group for 0.5-2.0 mg/mL of the test groups in the presence of S9 Mix. Based on the data obtained from this study, it was concluded that under the test condition, Everlan SL65 was positive effect in mammalian cell gene mutation test (in vitro).