Pyridinium, 1-[4-[[4-[(4-amino-9,10-dihydro-9,10-dioxo-3-sulfo-1-anthracenyl)amino]cyclohexyl]amino]-6-[(2,5-disulfophenyl)amino]-1,3,5-triazin-2-yl]-3-carboxy-, inner salt, sodium salt (1:3)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

CR SB08 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).

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 June 13, 2016 to August 23, 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:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

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:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

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:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

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:

cytotoxicity

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:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

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	130
	Positive control group^a	1329
	Test group
	5	153
	2.5	146
	1.25	148
	0.625	202
	0.3125	176
Without S9 Mix	Negative control group	121
	Positive control group	850
	Test group
	5	146
	2.5	127
	1.25	141
	0.625	135
	0.3125	134

^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	43	36	44 ± 9
	Positive control group^b	248	233	213	231 ± 18*
	Test group
	5	32	36	31	33 ± 3
	2.5	34	32	35	34 ± 2
	1.25	26	38	37	34 ± 7
	0.625	23	38	28	30 ± 8
	0.3125	33	32	34	33 ± 1
Without S9 Mix	Negative control group	40	23	23	29 ± 10
	Positive control group	359	312	326	332 ± 24*
	Test group
	5	21	24	26	24 ± 3
	2.5	28	26	30	28 ± 2
	1.25	29	24	26	26 ± 3
	0.625	21	24	21	22 ± 2
	0.3125	24	35	26	28 ± 6

^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	264	230	273	256 ± 23
	Positive control group^b	1152	1264	1215	1210 ± 56*
	Test group
	5	236	216	255	236 ± 20
	2.5	237	207	187	210 ± 25
	1.25	207	221	210	213 ± 7
	0.625	260	229	213	234 ± 24
	0.3125	216	266	283	255 ± 35
Without S9 Mix	Negative control group	271	228	252	250 ± 22
	Positive control group	741	726	802	756 ± 40*
	Test group
	5	201	223	197	207 ± 14
	2.5	231	222	192	215 ± 20
	1.25	217	250	217	228 ± 19
	0.625	249	241	208	233 ± 22
	0.3125	228	190	229	216 ± 22

^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	356	340	328	341 ± 14
	Positive control group^b	716	730	764	737 ± 25*
	Test group
	5	344	332	318	331 ± 14
	2.5	375	287	307	323 ± 46
	1.25	336	308	318	321 ± 14
	0.625	327	305	286	306 ± 21
	0.3125	343	321	371	345 ± 25
Without S9 Mix	Negative control group	335	370	355	353 ± 18
	Positive control group	782	831	903	839 ± 61*
	Test group
	5	302	328	334	321 ± 17
	2.5	356	307	370	344 ± 33
	1.25	341	284	302	309 ± 29
	0.625	368	326	307	334 ± 31
	0.3125	320	354	381	352 ± 31

^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	31	18	21	23 ± 7
	Positive control group^b	281	266	300	282 ± 17*
	Test group
	5	12	11	17	13 ± 3
	2.5	9	25	20	18 ± 8
	1.25	21	19	10	17 ± 6
	0.625	25	24	23	24 ± 1
	0.3125	22	15	17	18 ± 4
Without S9 Mix	Negative control group	33	31	23	29 ± 5
	Positive control group	263	277	244	261 ± 17*
	Test group
	5	28	22	33	28 ± 6
	2.5	18	21	25	21 ± 4
	1.25	19	26	27	24 ± 4
	0.625	28	19	24	24 ± 5
	0.3125	31	25	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	16	15	19	17 ± 2
	Positive control group^b	345	343	379	356 ± 20*
	Test group
	5	15	17	20	17 ± 3
	2.5	24	14	13	17 ± 6
	1.25	31	19	21	24 ± 6
	0.625	25	14	21	20 ± 6
	0.3125	17	19	22	19 ± 3
Without S9 Mix	Negative control group	17	8	16	14 ± 5
	Positive control group	618	709	775	701 ± 79*
	Test group
	5	16	15	18	16 ± 2
	2.5	18	16	21	18 ± 3
	1.25	22	27	22	24 ± 3
	0.625	20	18	22	20 ± 2
	0.3125	22	15	15	17 ± 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, CR SB08 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 M62-151100072001EN 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 SB08 show 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 CR SB08 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. No cytotoxicity was observed in all five tester strains up to 5mg/plate in the absence and presence of metabolite activations. Results showed that CR SB08 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, CR SB08 was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 5mg/plate in the absence and presence of S9 metabolic activation.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Based on the preliminary assay results, 5mg/plate was set as the highest dose in this study. In the mutagenicity assay, five doses of CR SB08 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. No cytotoxicity was observed in all five tester strains up to 5mg/plate in the absence and presence of metabolite activations. Results showed that CR SB08 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, CR SB08 was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 5mg/plate in the absence and presence of S9 metabolic activation.