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

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:

Sep. 20, 2016 - Dec 8, 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)

Version / remarks:

OECD 471:1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

305-729-0

EC Name:

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

Cas Number:

95009-01-1

Molecular formula:

C18H9CuN6O10S.Na

IUPAC Name:

Reaction products of diazotized 4-aminobenzenesulfonic acid coupled with resorcinol subsequently coupled with diazotised 2-amino-4,6-dinitrophenol, copper (2+) complexes, sodium salts

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): Everlan SL65
- Substance type: Powder
- Composition of test material, percentage of components: 71.19%
- Lot/batch No.: 3010
- 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

Details on mammalian cell type (if applicable):

Source of Cell: MOLTOX, molecular Toxicology, Inc.

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Controls

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).

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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

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	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).

Applicant's summary and conclusion

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.