5-({4-chloro-6-[(2-methylphenyl)amino]-1,3,5-triazin-2-yl}amino)-4-hydroxy-3-[(2-sulfophenyl)diazenyl]naphthalene-2,7-disulfonic acid, lithium sodium salts

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

CJ307 is not mutagenic. The result is read-across from Evercion SR61 in the reverse mutation analysis of Salmonellatyphimurium 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 October 03, 2016 to December 22, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The structure of the read-across substance, EC:300-504-3, is the same as the test substance. Therefore, the human toxicity and environmental toxicity is the same. The only difference is the test substance containing lithium. However, the low level of lithium will not change the classification.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Evercion SR61, Reactive red 3-1, EC: 300-504-3

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:

not determined

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 determined

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 determined

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 determined

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:

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	226
	Positive control group^a	1125
	Test group
	5	118
	2.5	171
	1.25	194
	0.625	200
	0.3125	207
Without S9 Mix	Negative control group	185
	Positive control group	657
	Test group
	5	160
	2.5	133
	1.25	179
	0.625	214
	0.3125	255

^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	42	32	49	41 ± 9
	Positive control group^b	284	314	344	314 ± 30*
	Test group
	5	55	44	53	51 ± 6
	2.5	44	65	42	50 ± 13
	1.25	42	42	33	39 ± 5
	0.625	54	49	53	52 ± 3
	0.3125	43	56	71	57 ± 14
Without S9 Mix	Negative control group	41	33	30	35 ± 6
	Positive control group	233	208	225	222 ± 13*
	Test group
	5	24	17	23	21 ± 4
	2.5	22	25	39	29 ± 9
	1.25	23	26	21	23 ± 3
	0.625	32	16	23	24 ± 8
	0.3125	30	40	26	32 ± 7

^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	219	295	205	240 ± 48
	Positive control group^b	1208	1429	1475	1371 ± 143*
	Test group
	5	203	195	218	205 ± 12
	2.5	237	247	197	227 ± 26
	1.25	194	200	196	197 ± 3
	0.625	159	257	235	217 ± 51
	0.3125	212	256	285	251 ± 37
Without S9 Mix	Negative control group	140	185	180	168 ± 25
	Positive control group	789	720	771	760 ± 36*
	Test group
	5	130	175	171	159 ± 25
	2.5	152	194	166	171 ± 21
	1.25	166	139	208	171 ± 35
	0.625	171	178	205	185 ± 18
	0.3125	186	154	223	188 ± 35

^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	434	392	422	416 ± 22
	Positive control group^b	897	881	911	896 ± 15*
	Test group
	5	443	520	460	474 ± 40
	2.5	425	437	443	435 ± 9
	1.25	461	465	439	455 ± 14
	0.625	460	379	425	421 ± 41
	0.3125	422	463	388	424 ± 38
Without S9 Mix	Negative control group	411	381	412	401 ± 18
	Positive control group	866	840	859	855 ± 13*
	Test group
	5	315	396	414	375 ± 53
	2.5	381	394	433	403 ± 27
	1.25	389	446	401	412 ± 30
	0.625	364	422	369	385 ± 32
	0.3125	435	397	402	411 ± 21

^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	19	25	27	24 ± 4
	Positive control group^b	276	307	300	294 ± 16*
	Test group
	5	29	25	19	24 ± 5
	2.5	23	20	16	20 ± 4
	1.25	14	19	24	19 ± 5
	0.625	28	18	16	21 ± 6
	0.3125	15	19	18	17 ± 2
Without S9 Mix	Negative control group	15	17	19	17 ± 2
	Positive control group	240	299	276	272 ± 30*
	Test group
	5	11	13	16	13 ± 3
	2.5	19	23	21	21 ± 2
	1.25	22	18	24	21 ± 3
	0.625	13	21	20	18 ± 4
	0.3125	21	19	18	19 ± 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: 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	9	12	13	11 ± 2
	Positive control group^b	417	451	409	426 ± 22*
	Test group
	5	10	8	15	11 ± 4
	2.5	9	15	14	13 ± 3
	1.25	14	17	13	15 ± 2
	0.625	17	11	14	14 ± 3
	0.3125	21	16	18	18 ± 3
Without S9 Mix	Negative control group	14	12	10	12 ± 2
	Positive control group	1019	947	1207	1058 ± 134*
	Test group
	5	10	12	10	11 ± 1
	2.5	8	8	15	10 ± 4
	1.25	7	9	10	9 ± 2
	0.625	14	9	9	11 ± 3
	0.3125	16	8	7	10 ± 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: 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, Evercion SR61 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 M62-151100096001EN 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 Evercion SR61 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 CJ303 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 Evercion SR61 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, Evercion SR61 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)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

CJ307 is not mutagenic. The result is read-across from Evercion SR61 in the reverse mutation analysis of Salmonellatyphimurium based on the similar structure. The experiment detail is described below.

Based on the preliminary assay results, 5mg/plate was set as the highest dose in this study. Inthe mutagenicity assay, five doses of Evercion SR61 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 Evercion SR61 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, Evercion SR61 was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 5mg/plate in the absence and presence of S9 metabolic activation.