Reaction products of diazotised Benzenesulfonic acid, 2-amino-5-[(2-hydroxyethyl)sulfonyl]- , subsequently coupled with 3,5-Diaminobenzoic acid, further coupled with diazotised Ethanol, 2-[(4-aminophenyl)sulfonyl]-, 1-(hydrogen sulfate), sodium salts

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

CR SB87 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 to July 25, 2016 October 07, 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	266
	Positive control group^a	1173
	Test group
	5	259
	2.5	302
	1.25	270
	0.625	258
	0.3125	243
Without S9 Mix	Negative control group	223
	Positive control group	796
	Test group
	5	275
	2.5	243
	1.25	280
	0.625	282
	0.3125	274

^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	34	30	38	34 ± 4
	Positive control group^b	262	243	236	247 ± 13*
	Test group
	5	25	35	26	29 ± 6
	2.5	24	25	29	26 ± 3
	1.25	29	28	34	30 ± 3
	0.625	32	35	31	33 ± 2
	0.3125	29	53	34	39 ± 13
Without S9 Mix	Negative control group	20	31	25	25 ± 6
	Positive control group	338	358	373	356 ± 18*
	Test group
	5	21	31	32	28 ± 6
	2.5	33	29	30	31 ± 2
	1.25	27	26	24	26 ± 2
	0.625	20	26	27	24 ± 4
	0.3125	26	24	32	27 ± 4

^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	255	240	237	244 ± 10
	Positive control group^b	1471	1308	1355	1378 ± 84*
	Test group
	5	339	211	293	281 ± 65
	2.5	278	295	255	276 ± 20
	1.25	230	275	228	244 ± 27
	0.625	219	279	240	246 ± 30
	0.3125	248	249	261	253 ± 7
Without S9 Mix	Negative control group	178	159	186	174 ± 14
	Positive control group	857	884	809	850 ± 38*
	Test group
	5	184	176	207	189 ± 16
	2.5	185	178	183	182 ± 4
	1.25	194	167	169	177 ± 15
	0.625	179	150	158	162 ± 15
	0.3125	188	185	166	180 ± 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 (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	407	397	436	413 ± 20
	Positive control group^b	1133	964	1054	1050 ± 85*
	Test group
	5	386	397	367	383 ± 15
	2.5	379	397	411	396 ± 16
	1.25	413	396	409	406 ± 9
	0.625	298	373	394	388 ± 12
	0.3125	468	376	409	418 ± 47
Without S9 Mix	Negative control group	369	419	385	391 ± 26
	Positive control group	1015	1071	912	999 ± 81*
	Test group
	5	343	341	356	347 ± 8
	2.5	407	392	368	389 ± 20
	1.25	384	371	341	365 ± 22
	0.625	320	371	374	355 ± 30
	0.3125	385	353	371	370 ± 16

^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	24	17	21	21 ± 4
	Positive control group^b	584	497	538	540 ± 44*
	Test group
	5	22	23	21	22 ± 1
	2.5	20	21	13	18 ± 4
	1.25	26	23	20	23 ± 3
	0.625	28	14	21	21 ± 7
	0.3125	19	18	21	19 ± 2
Without S9 Mix	Negative control group	17	18	20	18 ± 2
	Positive control group	238	261	237	245 ± 14*
	Test group
	5	18	16	12	15 ± 3
	2.5	23	18	14	18 ± 5
	1.25	19	27	19	22 ± 5
	0.625	27	20	19	22 ± 4
	0.3125	20	25	25	23 ± 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	12	15	14	14 ± 2
	Positive control group^b	296	289	295	293 ± 4*
	Test group
	5	7	10	19	12 ± 6
	2.5	10	19	13	14 ± 5
	1.25	12	7	15	11 ± 4
	0.625	13	10	16	13 ± 3
	0.3125	11	14	18	14 ± 4
Without S9 Mix	Negative control group	12	15	11	13 ± 2
	Positive control group	993	912	860	922 ± 67*
	Test group
	5	17	10	9	12 ± 4
	2.5	12	10	8	10 ± 2
	1.25	15	13	12	13 ± 2
	0.625	9	21	6	12 ± 8
	0.3125	12	16	16	15 ± 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: 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 SB87 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 M62-151100120001EN 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 SB87 show that test validity criteria was met.

Based on the preliminary assay results, 5mg/platewas set as the highest dose in this study. In the mutagenicity assay, five doses of CR SB87 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 SB87 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 SB87 was not mutagenic in the reverse mutation analysis ofSalmonellatyphimuriumup 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 SB87 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 SB87 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 SB87 was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 5mg/plate in the absence and presence of S9 metabolic activation.