Reaction products of diazotised Benzenesulfonic acid, 2-amino-5-[[2-(sulfooxy)ethyl]sulfonyl]-, subsequently coupled with Urea, N-(3-aminophenyl)-, subsequently condensation with 1,3,5-Triazine, 2,4,6-trichloro-, further condensation with Benzenesulfonic acid, 3-amino- and 3-Pyridinecarboxylic acid, sodium salts

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

CR SB07 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 27, 2016 to August 31, 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:

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 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	281
	Positive control group^a	1522
	Test group
	5	197
	2.5	248
	1.25	283
	0.625	229
	0.3125	224
Without S9 Mix	Negative control group	217
	Positive control group	691
	Test group
	5	213
	2.5	194
	1.25	236
	0.625	233
	0.3125	248

^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	35	40	31	35 ± 5
	Positive control group^b	320	337	359	339 ± 20*
	Test group
	5	32	29	32	31 ± 2
	2.5	39	26	51	39 ± 13
	1.25	44	34	35	38 ± 6
	0.625	30	40	20	30 ± 10
	0.3125	39	31	32	34 ± 4
Without S9 Mix	Negative control group	36	44	39	40 ± 4
	Positive control group	297	249	290	279 ± 26*
	Test group
	5	18	30	29	26 ± 7
	2.5	29	36	21	29 ± 8
	1.25	31	24	26	27 ± 4
	0.625	24	29	32	28 ± 4
	0.3125	26	36	43	35 ± 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	254	290	301	282 ± 25
	Positive control group^b	1264	1241	1322	1276 ± 42*
	Test group
	5	174	204	198	192 ± 16
	2.5	218	267	187	224 ± 40
	1.25	245	206	191	214 ± 28
	0.625	224	219	207	217 ± 9
	0.3125	274	269	234	259 ± 22
Without S9 Mix	Negative control group	228	263	219	237 ± 23
	Positive control group	656	686	734	692 ± 39*
	Test group
	5	235	222	188	215 ± 24
	2.5	219	262	233	238 ± 22
	1.25	215	229	251	232 ± 18
	0.625	247	224	276	249 ± 26
	0.3125	236	299	209	248 ± 46

^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	394	399	413	402 ± 10
	Positive control group^b	794	896	859	850 ± 52*
	Test group
	5	345	374	383	367 ± 20
	2.5	367	344	369	360 ± 14
	1.25	379	420	421	407 ± 24
	0.625	442	405	438	428 ± 20
	0.3125	437	411	377	408 ± 30
Without S9 Mix	Negative control group	356	419	382	386 ± 32
	Positive control group	856	828	860	848 ± 17*
	Test group
	5	374	341	349	355 ± 17
	2.5	409	350	374	378 ± 30
	1.25	326	368	356	350 ± 22
	0.625	369	387	351	369 ± 18
	0.3125	424	346	406	392 ± 41

^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	22	18	25	22 ± 4
	Positive control group^b	277	260	241	259 ± 18*
	Test group
	5	20	19	21	20 ± 1
	2.5	22	18	20	20 ± 2
	1.25	25	22	15	21 ± 5
	0.625	15	20	12	16 ± 4
	0.3125	19	21	26	22 ± 4
Without S9 Mix	Negative control group	28	18	26	24 ± 5
	Positive control group	217	220	225	221 ± 4*
	Test group
	5	19	27	19	22 ± 5
	2.5	30	32	24	29 ± 4
	1.25	26	24	14	21 ± 6
	0.625	23	24	23	23 ± 1
	0.3125	26	30	30	29 ± 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	12	10	20	14 ± 5
	Positive control group^b	239	291	353	294 ± 57*
	Test group
	5	8	10	13	10 ± 3
	2.5	13	10	11	11 ± 2
	1.25	14	16	19	16 ± 3
	0.625	16	12	21	16 ± 5
	0.3125	15	13	14	14 ± 1
Without S9 Mix	Negative control group	5	13	10	9 ± 4
	Positive control group	712	828	725	755 ± 64*
	Test group
	5	16	19	14	16 ± 3
	2.5	11	18	15	15 ± 4
	1.25	14	14	22	17 ± 5
	0.625	12	14	14	13 ± 1
	0.3125	13	11	11	12 ± 1

^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 SB07 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 SB07 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 SB07 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 toxicity was observed in all five tester strains up to 5mg/plate in the absence and presence of metabolite activations. Results showed that CR SB07 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 SB07 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

Genetic toxicity in vitro

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 SB07 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 toxicity was observed in all five tester strains up to 5 mg/plate in the absence and presence of metabolite activations. Results showed that CR SB07 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 SB07 was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 5mg/plate in the absence and presence of S9 metabolic activation.