Pyridinium, 1-[2-[[4-[[3-[2-[6-amino-1-hydroxy-3-sulfo-5-[2-[2-sulfo-4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]diazenyl]-2-naphthalenyl]diazenyl]-4-sulfophenyl]amino]-4-oxobutyl]sulfonyl]ethyl]-3-carboxy-, inner salt, sodium salt (1:4)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

CR SB33 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 (OECD TG471).

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From January 4, 2019 to September 16, 2019

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

Test concentrations with justification for top dose:

Range-finding test showed that article without rat S9 Mix and without hamster S9 Mix at the dose of 5 mg/plate did not cause toxicity to all tested strains. Therefore, the highest dose on definitive test was 5 mg/plate, other four doses were 2.5, 1.25, 0.625 and 0.3125 mg/plate, respectively.

Negative solvent / vehicle controls:

yes

Remarks:

Reverse osmosis water

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-Aminoanthracene

Details on test system and experimental conditions:

Culture medium: Nutrient broth (NB No.2).
Incubation condition: 35±1℃

Evaluation criteria:

1. Positive response criteria: The test chemical was considered to be positively reactive if any of the following criteria was fulfilled.
1.a The colony number of at least one of the dose groups was higher two folds than negative control group, and calculated by statistic software SPSS, p<0.05 (Duncan's multiple range test of One-Way ANOVA).
1.b The colony number of at least one of the dose groups was higher two folds than negative control group, and presented dose-reponse relaationship.
2. Negative response criterion: The test results for each test group of test article failed to comply with the positive response criteria were judged the negative response.

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

Table 1. Toxicity of the test article ofSalmonella typhimurium(without rat S9 Mix)

Group		Control article/Test article^a		Colony number (CFU/plate)
				TA98		TA100		TA102		TA1535		TA1537
				1	2	1	2	1	2	1	2	1	2
Without rat S9 Mix		Negative control article		32	29	157	167	411	408	35	33	15	14
		Positive control article		281	328	716	783	959	932	201	200	213	211
		Test group (mg/plate)
		5		27	31	163	166	425	409	31	26	18	6
		2.5		33	21	148	154	407	432	40	29	19	9
		1.25		29	28	144	155	405	442	40	29	9	14
		0.625		31	21	168	162	401	435	39	30	9	14
		0.3125		35	28	162	161	425	408	37	30	18	11

^aNegative control article: Reverse osmosis water; Positive control article: 4 -Nitroquinoline-1-oxide (0.5 μg/plate), Sodium azide (0.4, 5.0

μg/plate), Mitomycin C (0.5 μg/plate) and 9 -Aminiacridine (50.0 μg/plate).

Table 2. Toxicity of the test article ofSalmonella typhimurium(without hamster S9 Mix)

Group		Control article/Test article^a		Colony number (CFU/plate)
				TA98		TA100		TA102		TA1535		TA1537
				1	2	1	2	1	2	1	2	1	2
Without hamster S9 Mix		Negative control article		32	31	183	172	298	320	26	20	11	9
		Positive control article		229	306	624	589	805	812	196	175	489	449
		Test group (mg/plate)
		5		22	27	142	176	326	296	23	13	9	14
		2.5		25	31	159	171	342	332	20	17	11	9
		1.25		30	32	183	187	299	306	20	17	5	15
		0.625		28	29	183	177	330	316	24	15	10	12
		0.3125		33	31	199	176	308	349	15	20	15	6

^aNegative control article: Reverse osmosis water; Positive control article: 4 -Nitroquinoline-1-oxide (0.5 μg/plate), Sodium azide (0.4, 5.0

μg/plate), Mitomycin C (0.5 μg/plate) and 9 -Aminiacridine (50.0 μg/plate).

Table 3. Reverse mutation test ofSalmonella typhimuriumTA98 (with and without rat S9 Mix)

Group	Control article/Test article^a	Colony number (CFU/plate)			Average of colonies^b
		1	2	3
With rat S9 Mix	Negative control group	29	23	33	28 ± 5
	Positive control group	244	265	238	249 ± 14*
	Test group (mg/plate)
	5	28	28	22	26 ± 3
	2.5	31	29	27	29 ± 2
	1.25	23	36	20	26 ± 9
	0.625	24	21	30	25 ± 5
	0.3125	25	25	24	25 ± 1
Without rat S9 Mix	Negative control group	39	34	31	35 ± 4
	Positive control group	298	314	302	305 ± 8*
	Test group (mg/plate)
	5	20	25	39	28 ± 10
	2.5	32	34	40	35 ± 4
	1.25	25	36	28	30 ± 6
	0.625	27	37	26	30 ± 6
	0.3125	30	26	35	30 ± 5

^aNegative control article: Reverse osmosis water; Positive control group: Benzo[a]pyrene (4.0 μg/plate) in S9 activation group and 4 -Nitroquinoline-1-oxide (0.5 μg/plate) in without S9 activation group.

^bAverage of colonies was shown as Mean ± SD, the data were triplicate.

*Reverse mutant colony number were twice more than negative control group or p<0.05 (Duncan's multiple range test of One-Way ANOVA).

Table 4. Reverse mutation test ofSalmonella typhimuriumTA98 (with and without hamster S9 Mix)

Group	Control article/Test article^a	Colony number (CFU/plate)			Average of colonies^b
		1	2	3
With hamster S9 Mix	Negative control group	32	39	38	36 ± 4
	Positive control group	316	305	318	313 ± 7*
	Test group (mg/plate)
	5	44	42	35	40 ± 5
	2.5	42	31	38	37 ± 6
	1.25	30	36	39	35 ± 5
	0.625	36	30	38	35 ± 4
	0.3125	32	42	34	36 ± 5
Without hamster S9 Mix	Negative control group	30	25	23	26 ± 4
	Positive control group	218	258	274	250 ± 29*
	Test group (mg/plate)
	5	39	28	35	34 ± 6
	2.5	25	23	25	24 ± 1
	1.25	34	30	30	31 ± 2
	0.625	22	30	29	27 ± 4
	0.3125	28	26	22	25 ± 3

^aNegative control article: Reverse osmosis water; Positive control group: Benzo[a]pyrene (4.0 μg/plate) in S9 activation group and 4 -Nitroquinoline-1-oxide (0.5 μg/plate) in without S9 activation group.

^bAverage of colonies was shown as Mean ± SD, the data were triplicate.

*Reverse mutant colony number were twice more than negative control group or p<0.05 (Duncan's multiple range test of One-Way ANOVA).

Table 5. Reverse mutation test ofSalmonella typhimuriumTA100 (with and without rat S9 Mix)

Group	Control article/Test article^a	Colony number (CFU/plate)			Average of colonies^b
		1	2	3
With rat S9 Mix	Negative control group	202	209	210	207 ± 4
	Positive control group	692	634	708	678 ± 39*
	Test group (mg/plate)
	5	200	194	211	202 ± 9
	2.5	203	202	202	202 ± 1
	1.25	205	193	213	204 ± 10
	0.625	213	194	205	204 ± 10
	0.3125	198	217	205	207 ± 10
Without rat S9 Mix	Negative control group	167	180	178	175 ± 7
	Positive control group	759	765	752	759 ± 7*
	Test group (mg/plate)
	5	182	180	175	179 ± 4
	2.5	185	187	173	182 ± 8
	1.25	176	171	183	177 ± 6
	0.625	171	180	178	176 ± 5
	0.3125	172	192	186	183 ± 10

^aNegative control article: Reverse osmosis water; Positive control group: 2-Aminoanthracene (4.0 μg/plate) in S9 activation group and Sodium azide (5.0 μg/plate) in without S9 activation group.

^bAverage of colonies was shown as Mean ± SD, the data were triplicate.

*Reverse mutant colony number were twice more than negative control group or p<0.05 (Duncan's multiple range test of One-Way ANOVA).

Table 6. Reverse mutation test ofSalmonella typhimuriumTA100 (with and without hamster S9 Mix)

Group	Control article/Test article^a	Colony number (CFU/plate)			Average of colonies^b
		1	2	3
With hamster S9 Mix	Negative control group	208	189	181	193 ± 14
	Positive control group	740	760	795	765 ± 28*
	Test group (mg/plate)
	5	197	185	182	188 ± 8
	2.5	184	184	171	180 ± 8
	1.25	179	185	169	178 ± 8
	0.625	187	182	187	185 ± 3
	0.3125	195	169	196	187 ± 15
Without hamster S9 Mix	Negative control group	198	185	183	189 ± 8
	Positive control group	601	642	622	622 ± 21*
	Test group (mg/plate)
	5	198	172	186	185 ± 13
	2.5	183	183	194	187 ± 6
	1.25	177	198	185	187 ± 11
	0.625	180	176	177	178 ± 2
	0.3125	185	195	173	184 ± 11

^aNegative control article: Reverse osmosis water; Positive control group: 2-Aminoanthracene (4.0 μg/plate) in S9 activation group and Sodium azide (5.0 μg/plate) in without S9 activation group.

^bAverage of colonies was shown as Mean ± SD, the data were triplicate.

*Reverse mutant colony number were twice more than negative control group or p<0.05 (Duncan's multiple range test of One-Way ANOVA).

Table 7. Reverse mutation test ofSalmonella typhimuriumTA102 (with and without rat S9 Mix)

Group	Control article/Test article^a	Colony number (CFU/plate)			Average of colonies^b
		1	2	3
With rat S9 Mix	Negative control group	417	390	398	402 ± 14
	Positive control group	989	998	995	994 ± 5*
	Test group (mg/plate)
	5	396	387	402	395 ± 8
	2.5	402	392	405	400 ± 7
	1.25	418	413	395	409 ± 12
	0.625	412	414	386	404 ± 16
	0.3125	414	415	409	413 ± 3
Without rat S9 Mix	Negative control group	395	385	384	388 ± 6
	Positive control group	994	982	1093	1023 ± 61*
	Test group (mg/plate)
	5	351	348	396	365 ± 27
	2.5	414	380	389	394 ± 18
	1.25	386	373	398	386 ± 13
	0.625	375	392	413	393 ± 19
	0.3125	368	382	403	384 ± 18

^aNegative control article: Reverse osmosis water; Positive control group: 2-Aminoanthracene (10.0 μg/plate) in S9 activation group and Mitomycin C (0.5 μg/plate) in without S9 activation group.

^bAverage of colonies was shown as Mean ± SD, the data were triplicate.

*Reverse mutant colony number were twice more than negative control group or p<0.05 (Duncan's multiple range test of One-Way ANOVA).

Table 8. Reverse mutation test ofSalmonella typhimuriumTA102 (with and without hamster S9 Mix)

Group	Control article/Test article^a	Colony number (CFU/plate)			Average of colonies^b
		1	2	3
With hamster S9 Mix	Negative control group	427	431	420	426 ± 6
	Positive control group	1085	1084	1094	1088 ± 6*
	Test group (mg/plate)
	5	405	445	434	428 ± 21
	2.5	457	425	428	437 ± 18
	1.25	435	429	459	441 ± 16
	0.625	426	422	473	440 ± 28
	0.3125	428	446	439	438 ± 9
Without hamster S9 Mix	Negative control group	315	298	326	313 ± 14
	Positive control group	949	920	1010	960 ± 46*
	Test group (mg/plate)
	5	312	333	316	320 ± 11
	2.5	306	287	293	295 ± 10
	1.25	303	340	329	324 ± 19
	0.625	317	357	327	334 ± 21
	0.3125	293	303	294	297 ± 6

^aNegative control article: Reverse osmosis water; Positive control group: 2-Aminoanthracene (10.0 μg/plate) in S9 activation group and Mitomycin C (0.5 μg/plate) in without S9 activation group.

^bAverage of colonies was shown as Mean ± SD, the data were triplicate.

*Reverse mutant colony number were twice more than negative control group or p<0.05 (Duncan's multiple range test of One-Way ANOVA).

Table 9. Reverse mutation test ofSalmonella typhimuriumTA1535 (with and without rat S9 Mix)

Group	Control article/Test article^a	Colony number (CFU/plate)			Average of colonies^b
		1	2	3
With rat S9 Mix	Negative control group	16	24	21	20 ± 4
	Positive control group	184	205	206	198 ± 12*
	Test group (mg/plate)
	5	17	31	22	23 ± 7
	2.5	28	25	25	26 ± 2
	1.25	22	22	38	27 ± 9
	0.625	15	23	22	20 ± 4
	0.3125	22	21	24	22 ± 2
Without rat S9 Mix	Negative control group	22	32	24	26 ± 5
	Positive control group	238	234	228	233 ± 5*
	Test group (mg/plate)
	5	25	29	20	25 ± 5
	2.5	23	27	24	25 ± 2
	1.25	25	21	29	25 ± 4
	0.625	28	32	31	30 ± 2
	0.3125	31	25	24	27 ± 4

^aNegative control article: Reverse osmosis water; Positive control group: 2-Aminoanthracene (4.0 μg/plate) in S9 activation group and Sodium azide (0.4 μg/plate) in without S9 activation group.

^bAverage of colonies was shown as Mean ± SD, the data were triplicate.

*Reverse mutant colony number were twice more than negative control group or p<0.05 (Duncan's multiple range test of One-Way ANOVA).

Table 10. Reverse mutation test ofSalmonella typhimuriumTA1535 (with and without hamster S9 Mix)

Group	Control article/Test article^a	Colony number (CFU/plate)			Average of colonies^b
		1	2	3
With   hamster S9 Mix	Negative control group	21	11	17	16 ± 5
	Positive control group	247	273	253	258 ± 14*
	Test group (mg/plate)
	5	20	21	18	20 ± 2
	2.5	20	17	23	20 ± 3
	1.25	17	22	15	18 ± 4
	0.625	19	19	27	22 ± 5
	0.3125	24	17	20	20 ± 4
Without   hamster S9 Mix	Negative control group	18	20	22	20 ± 2
	Positive control group	228	245	218	230 ± 14*
	Test group (mg/plate)
	5	14	22	15	17 ± 4
	2.5	22	16	17	18 ± 3
	1.25	17	19	22	19 ± 3
	0.625	21	18	22	20 ± 2
	0.3125	19	20	18	19 ± 1

^aNegative control article: Reverse osmosis water; Positive control group: 2-Aminoanthracene (4.0 μg/plate) in S9 activation group and Sodium azide (0.4 μg/plate) in without S9 activation group.

^bAverage of colonies was shown as Mean ± SD, the data were triplicate.

*Reverse mutant colony number were twice more than negative control group or p<0.05 (Duncan's multiple range test of One-Way ANOVA).

Table 11. Reverse mutation test ofSalmonella typhimuriumTA1537 (with and without rat S9 Mix)

Group	Control article/Test article^a	Colony number (CFU/plate)			Average of colonies^b
		1	2	3
With rat S9 Mix	Negative control group	14	7	11	11 ± 4
	Positive control group	261	241	281	261 ± 20*
	Test group (mg/plate)
	5	10	13	9	11 ± 2
	2.5	17	10	11	13 ± 4
	1.25	11	13	7	10 ± 3
	0.625	15	12	7	11 ± 4
	0.3125	12	11	8	10 ± 2
Without rat S9 Mix	Negative control group	12	11	9	11 ± 2
	Positive control group	216	289	274	260 ± 39*
	Test group (mg/plate)
	5	9	12	18	13 ± 5
	2.5	8	9	12	10 ± 2
	1.25	7	6	15	9 ± 5
	0.625	7	19	8	11 ± 7
	0.3125	11	6	9	9 ± 3

^aNegative control article: Reverse osmosis water; Positive control group: 2-Aminoanthracene (4.0 μg/plate) in S9 activation group and 9-Aminoacridine (50.0 μg/plate) in without S9 activation group.

^bAverage of colonies was shown as Mean ± SD, the data were triplicate.

*Reverse mutant colony number were twice more than negative control group or p<0.05 (Duncan's multiple range test of One-Way ANOVA).

Table 12. Reverse mutation test ofSalmonella typhimuriumTA1537 (with and without hamster S9 Mix)

Group	Control article/Test article^a	Colony number (CFU/plate)			Average of colonies^b
		1	2	3
With   hamster S9 Mix	Negative control group	14	15	10	13 ± 3
	Positive control group	472	480	457	470 ± 12*
	Test group (mg/plate)
	5	16	12	15	14 ± 2
	2.5	13	10	11	11 ± 2
	1.25	17	20	13	17 ± 4
	0.625	15	17	19	17 ± 2
	0.3125	17	13	22	17 ± 5
Without   hamster S9 Mix	Negative control group	15	17	13	15 ± 2
	Positive control group	308	329	355	331 ± 24*
	Test group (mg/plate)
	5	12	20	10	14 ± 5
	2.5	16	22	17	18 ± 3
	1.25	17	15	21	18 ± 3
	0.625	17	16	13	15 ± 2
	0.3125	17	12	20	16 ± 4

^aNegative control article: Reverse osmosis water; Positive control group: 2-Aminoanthracene (4.0 μg/plate) in S9 activation group and 9-Aminoacridine (50.0 μg/plate) in without S9 activation group.

^bAverage of colonies was shown as Mean ± SD, the data were triplicate.

*Reverse mutant colony number were twice more than negative control group or p<0.05 (Duncan's multiple range test of One-Way ANOVA).

Conclusions:

According to OECD 471 test method, CR SB33 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 MZ6-181200025 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 SB33 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 SB33 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 or hamster S9 Mix activation. No cytotoxicity was observed in all five tester strains up to 5 mg/plate in the absence and presence of metabolite activations. Results showed that CR SB33 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 SB33 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

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 SB33 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. No cytotoxicity was observed in all five tester strains up to 5 mg/plate in the absence and presence of metabolite activations. Results showed that CR SB33 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 SB33 was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 5mg/plate in the absence and presence of S9 metabolic activation.