2,7-Naphthalenedisulfonic acid, 4-amino-5-hydroxy-3-[2-[3-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]diazenyl]-6-[2-[2-sulfo-4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]diazenyl]-, sodium salt (1:5)

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 October 16, 2017 to December 22, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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:

2,7-Naphthalenedisulfonic acid, 4-amino-5-hydroxy-3-[2-[3-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]diazenyl]-6-[2-[2-sulfo-4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]diazenyl]-, sodium salt (1:5)

EC Number:

815-135-8

Cas Number:

1386899-40-6

Molecular formula:

C26H25N5O22S7.5Na

IUPAC Name:

2,7-Naphthalenedisulfonic acid, 4-amino-5-hydroxy-3-[2-[3-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]diazenyl]-6-[2-[2-sulfo-4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]diazenyl]-, sodium salt (1:5)

Test material form:

solid: particulate/powder

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:

Rat liver S9 mix and hamster liver 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 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

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 3

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 4

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 5

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 6

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	R plasmid^a
TA98	+	+	+	+
TA100	+	+	+	+
TA102	+	ϴ	+	+
TA1535	+	+	+	ϴ
TA1537	+	+	+	ϴ

^a R plasmid: Ampicillin resistance were used for R plasmid test.

+ 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)			Average of colonies^a
		1	2	3
Without S9 activation	Negative control group	182	185	199	189 ± 9
	Positive control group^b	854	980	845	893±75
	Test group
	5	180	201	183	188 ± 11
	2.5	173	184	192	183 ± 10
	1.25	199	180	172	184 ± 14
	0.625	185	195	184	188 ± 6
	0.3125	192	204	183	193 ± 11

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

^b Positive control group: 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
Rat liver S9 activation	Negative control group	37	31	33	34 ± 3
	Positive control group^b	256	199	218	224 ± 29*
	Test group
	5	38	39	39	39 ± 1
	2.5	34	38	39	37 ± 3
	1.25	31	39	37	36 ± 4
	0.625	43	36	30	36 ± 7
	0.3125	43	48	33	41 ± 8
Hamster liver S9 activation	Negative control group	36	32	38	35 ± 3
	Positive control group	201	270	235	235 ± 35*
	Test group
	5	31	34	35	33 ± 2
	2.5	31	26	36	31 ± 5
	1.25	41	30	41	37 ± 6
	0.625	40	42	43	42 ± 2
	0.3125	41	48	45	45 ± 4
Without S9 activation	Negative control group	31	33	39	34 ± 4
	Positive control group	205	249	240	231 ± 23*
	Test group
	5	31	36	34	34 ± 3
	2.5	30	36	42	36 ± 6
	1.25	30	35	36	34 ± 3
	0.625	33	34	36	34 ± 2
	0.3125	35	36	35	35 ± 1

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

^b Positive control group:

Rat liver S9 activation and Hamster liver S9 activation group: Benzo [a] pyrene (4.0μg/plate).

Without S9 activation group: 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
Rat liver S9 activation	Negative control group	185	165	182	177 ± 11
	Positive control group^b	662	683	608	651 ± 39*
	Test group
	5	173	175	177	175 ± 2
	2.5	173	179	172	175 ± 4
	1.25	178	182	183	181 ± 3
	0.625	171	193	182	182 ± 11
	0.3125	198	184	182	188 ± 9
Hamster liver S9 activation	Negative control group	175	167	173	172 ± 4
	Positive control group	684	614	699	666 ± 45*
	Test group
	5	182	183	186	184 ± 2
	2.5	184	188	176	183 ± 6
	1.25	176	171	180	176 ± 5
	0.625	170	174	196	180 ± 14
	0.3125	177	173	178	176 ± 3
Without S9 activation	Negative control group	163	168	173	168 ± 5
	Positive control group	675	579	606	620 ± 50*
	Test group
	5	164	162	177	168 ± 8
	2.5	178	168	158	168 ± 10
	1.25	178	176	167	174 ± 6
	0.625	167	187	182	179 ± 10
	0.3125	180	165	160	168 ± 10

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

^b Positive control group:

Rat liver S9 activation and Hamster liver S9 activation group:2-Aminoanthracene (4.0μg/plate).

Without S9 activation group: 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
Rat liver S9 activation	Negative control group	438	436	447	440 ± 6
	Positive control group^b	871	939	901	904 ± 34*
	Test group
	5	441	445	466	451 ± 13
	2.5	472	465	464	467 ± 4*
	1.25	429	432	419	427 ± 7
	0.625	425	431	424	427 ± 4
	0.3125	433	442	438	438 ± 5
Hamster liver S9 activation	Negative control group	405	412	441	419 ± 19
	Positive control group	896	870	830	865 ± 33*
	Test group
	5	448	419	441	436 ± 15
	2.5	425	446	452	441 ± 14
	1.25	455	448	439	447 ± 8
	0.625	425	442	442	436 ± 10
	0.3125	462	452	442	452 ± 10
Without S9 activation	Negative control group	399	383	387	390 ± 8
	Positive control group	840	972	870	894 ± 69*
	Test group
	5	423	433	422	426 ± 6
	2.5	454	457	435	449 ± 12*
	1.25	420	449	433	434 ± 15
	0.625	413	428	417	419 ± 8
	0.3125	429	462	448	446 ± 17*

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

^b Positive control group:

Rat liver S9 activation and Hamster liver S9 activation group: 2-Aminoanthracene (10.0μg/plate).

Without S9 activation group: 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
Rat liver S9 activation	Negative control group	17	17	15	16 ± 1
	Positive control group^b	356	482	286	375 ± 99*
	Test group
	5	9	13	10	11 ± 2
	2.5	7	13	12	11 ± 3
	1.25	18	17	12	16 ± 3
	0.625	16	17	13	15 ± 2
	0.3125	15	19	16	17 ± 2
Hamster liver S9 activation	Negative control group	20	29	27	25 ± 5
	Positive control group	250	240	232	241 ± 9*
	Test group
	5	21	18	22	20 ± 2
	2.5	17	32	22	24 ± 8
	1.25	22	21	23	22 ± 1
	0.625	23	15	22	20 ± 4
	0.3125	17	23	22	21 ± 3
Without S9 activation	Negative control group	15	15	18	16 ± 2
	Positive control group	204	199	271	225 ± 40*
	Test group
	5	15	19	18	17 ± 2
	2.5	12	11	8	10 ± 2
	1.25	13	18	10	14 ± 4
	0.625	8	10	10	9 ± 1
	0.3125	14	14	12	13 ± 1

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

^b Positive control group:

Rat liver S9 activation and Hamster liver S9 activation group:2-Aminoanthracene (4.0μg/plate).

Without S9 activation group: 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
Rat liver S9 activation	Negative control group	10	10	9	10 ± 1
	Positive control group^b	425	538	344	436 ± 97*
	Test group
	5	7	8	9	8 ± 1
	2.5	8	6	13	9 ± 4
	1.25	7	6	10	8 ± 2
	0.625	9	8	13	10 ± 3
	0.3125	7	8	13	9 ± 3
Hamster liver S9 activation	Negative control group	15	14	10	13 ± 3
	Positive control group	431	441	455	442 ± 12*
	Test group
	5	10	10	9	10 ± 1
	2.5	8	10	13	10 ± 3
	1.25	8	9	10	9 ± 1
	0.625	10	9	12	10 ± 2
	0.3125	10	9	11	10 ± 1
Without S9 activation	Negative control group	10	7	10	9 ± 2
	Positive control group	312	336	358	335 ± 23*
	Test group
	5	10	6	6	7 ± 2
	2.5	11	12	8	10 ± 2
	1.25	9	12	9	10 ± 2
	0.625	10	8	9	9 ± 1
	0.3125	8	4	8	7 ± 2

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

^b Positive control group:

Rat liver S9 activation and Hamster liver S9 activation group: 2-Aminoanthracene (4.0μg/plate).

Without S9 activation group: 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, CR SB37 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 M62-170900011001EN 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 CR SB37 show that test validity criteria was met.

Based on the preliminary assay results, 5 mg/plate was set as the highest dose in this study. In the mutagenicity assay, five doses of CR SB37 at 0.3125, 0.625, 1.25, 2.5 and 5 mg/plate, negative control and positive controls were tested in tester strains TA98, TA100, TA102, TA1535 and TA1537 in triplicate with or without S9 mix activation. The metabolite activations were rat liver S9 mix activation and hamster liver S9 mix activation. Results showed that CR SB37 did not increase the number of revertants in all five tester strains TA98, TA100, TA102, TA1535 and TA1537 up to 5 mg/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, CR SB37 was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 5 mg/plate in the absence and presence of S9 metabolic activation.