N,N’-di[3-(p-toluene sulfonyl)oxy]phenyl urea

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

July 21, 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N,N’-di[3-(p-toluene sulfonyl)oxy]phenyl urea

EC Number:

876-151-9

Cas Number:

2292123-68-1

Molecular formula:

C27H24N2O7S2

IUPAC Name:

N,N’-di[3-(p-toluene sulfonyl)oxy]phenyl urea

Test material form:

solid: particulate/powder

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9 mix

Test concentrations with justification for top dose:

Main test -1 (-/+S9 mix): 4.88, 19.5, 78.1, 313, 1250, 5000 µg/plate.
Main test -2 (-/+ S9 mix): 313, 625, 1250, 2500, 5000 µg/plate.
5000µg/plate selected as the highest dose as precipitation of the test item was observed.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: The test item was insoluble in distilled water at 50 mg/mL. Test item solution of 50 mg/mL was prepared in DMSO. The test item was soluble and considered to be stable from the observations; no colour change, gas generation, etc. Dehydrated DMSO was selected as vehicle as the test item can hydrolyse with strong alkali.

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

furylfuramide

other:

Remarks:

5 positive controls are used

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- preincubation; incorporation in agar and poured on to agar plate

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 20 minutes at 37°C +/- 0.5°C
- Exposure duration/duration of treatment: Incubation period = 48 hours
- Number of revertant colonies counted after incubation period

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Any supplementary information relevant to cytotoxicity: none observed

Rationale for test conditions:

As required by the OECD guideline

Evaluation criteria:

- Test item was judged to be positive when the number of revertant colonies increased to twice or more than the negative control and when the responses were dose-related and/or reproducible. The other cases were judged to be negative.

Statistics:

No statistical methods used

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Any other information on results incl. tables

Table 2: Main test 1

Test period	From July 26, 2018 to July 30, 2018
With (+) or Without (-) S9 mix	Test item dose (µg/plate)	Number of revertant colonies per plate
		Base-pair substitution						Frameshift
		TA100		TA1535		WP2uvrA		TA98		TA1537
-S9 mix	Negative control	89	(90± 7)	10	(9 ± 2)	20	(24 ± 8)	18	(18 ± 4)	6	(12 ± 6)
		83		7		19		15		12
		97		10		34		22		18
	4.88	91	(96 ± 10)	11	(10 ± 2)	25	(21 ± 4)	14	(17 ± 7)	13	(16 ± 4)
		89		10		18		11		21
		107		8		19		25		15
	19.5	100	(93 ± 6	7	(6 ± 1)	15	(18 ± 5)	20	(17 ± 5)	15	(13 ± 4)
		90		7		15		12		8
		89		5		24		20		15
	78.1	87	(85 ± 6)	10	(8 ± 3)	34	(25 ± 8)	20	(15 ± 6)	14	(13 ± 2)
		90		4		18		8		11
		89		10		22		18		14
	313*	93	(88 ± 13)	3	(4 ± 4)	24	(20 ± 4)	15	(14 ± 1)	17	(12 ± 4)
		98		8		20		13		10
		74		1		17		13		10
	1250*	76	(82 ± 8)	4	(5 ± 1)	10	(14 ± 4)	11	(10 ± 2)	8	(8 ± 1)
		79		6		18		8		7
		91		5		14		11		9
	5000*	77	(77 ± 9)	8	(7 ± 1)	28	(22 ± 6)	8	(11 ± 3)	13	(11 ± 2)
		68		6		16		12		9
		85		6		23		14		11
+S9 mix	Negative control	90	(89 ± 2)	6	(6 ± 2)	21	(26 ± 6)	25	(22 ± 3)	17	(17 ± 5)
		87		4		24		21		12
		91		7		32		20		22
	4.88	90	(95 ± 5)	7	(10 ± 6)	29	(28 ± 4)	22	(30 ± 7)	10	(14 ± 5)
		94		7		24		36		13
		100		17		32		33		19
	19.5	93	(90 ± 25)	10	(11 ± 1)	22	(27 ± 4)	24	( 30 ± 7)	19	(21 ± 4)
		85		11		29		29		25
		85		12		29		38		18
	78.1	119	(89 ± 11)	8	(8 ± 4)	33	(32 ± 4)	26	(28 ± 7)	11	(14 ± 3)
		78		5		27		22		17
		74		12		35		36		13
	313*	83	(89 ± 2)	6	(9 ± 3)	20	(23 ± 3)	32	( 29 ± 7)	13	(15 ± 3)
		83		9		22		21		15
		102		11		26		34		18
	1250*	87	(86 ± 6)	4	(7 ± 4)	21	(22 ± 2)	26	(22 ± 4)	11	(11 ± 1)
		91		5		25		21		11
		89		11		21		19		10
	5000*	89	(86 ± 6)	6	(6 ± 2)	17	(15 ± 2)	24	(23 ± 3)	12	(13 ± 2)
		79		4		14		25		15
		91		7		14		19		13
Positive control -S9 mix	Chemical	AF-2		NaN3		AF-2		AF-2		ICR-191
	Dose ((µg/plate)	0.01		0.5		0.01		0.1		0.5
	No. of revertant colonies/plate	680	(692 ± 12)	492	(492 ± 11)	122	(120 ± 8)	488	(498 ± 9)	474	(482 ± 29)
		703		503		111		504		457
		692		481		127		503		514
Positive control +S9 mix	Chemical	2AA		2AA		2AA		B[a]P		2AA
	Dose ((µg/plate)	1		2		10		5		2
	No. of revertant colonies/plate	897	(896 ± 3)	191	(179 ± 11)	476	(449 ± 56)	318	(302 ± 17)	167	(177 ± 25)
		899		169		485		284		206
		893		177		385		303		159

Note: ( ) The mean number of revertant colonies per plate ± standard deviation (n=3)

* Precipitation of the test item was observed in all plates for each tester strain

AF-2: furylfuramide

NaN3: Sodium azide

2AA: 2-aminoanthracene

B[a]P: Benzo[a]pyrene 

 

Table 3: Main test 2

Test period	From August 10, 2018 to August 13, 2018
With (+) or Without (-) S9 mix	Test item dose (µg/plate)	Number of revertant colonies per plate
		Base-pair substitution						Frameshift
		TA100		TA1535		WP2uvrA		TA98		TA1537
-S9 mix	Negative control	99	(94± 4)	8	(7 ± 1)	39	(44 ± 5)	29	(23 ± 10)	6	(11 ± 6)
		93		6		48		11		11
		91		8		45		28		17
	313*	83	(76 ± 11)	8	(8 ± 3)	32	(35 ± 3)	17	(14 ± 3)	7	(8 ± 3)
		81		5		37		14		6
		63		11		36		11		11
	625*	62	(75 ± 12)	5	(6 ± 3)	37	(29 ± 8)	14	(11 ± 3)	8	(9 ± 3)
		82		9		28		11		12
		82		4		21		8		7
	1250*	71	(77 ± 9)	8	(8 ± 2)	36	(34 ± 2)	8	(12 ± 5)	10	(7 ± 4)
		87		9		32		11		9
		74		6		35		17		3
	2500*	74	(79 ± 4)	6	(5 ± 2)	42	(37 ± 5)	15	(13 ± 2)	4	(4 ± 1)
		80		2		35		13		5
		78		6		33		12		4
	5000*	87	(82 ± 5)	7	(8 ± 3)	42	(38 ± 7)	15	(13 ± 2)	5	(4 ± 1)
		80		12		30		11		4
		78		6		42		14		4
+S9 mix	Negative control	85	(88 ± 9)	7	(14 ± 7)	29	(40 ± 14)	26	(26 ± 6)	10	(13 ± 4)
		80		20		55		21		18
		98		15		35		32		12
	313*	72	(73 ± 5)	5	(6 ± 3)	33	(37 ± 4)	21	(27 ± 6)	15	(18 ± 4)
		78		3		37		27		23
		69		9		41		33		17
	625*	77	(81 ± 3)	14	(8 ± 6)	28	(35 ± 7)	29	(32 ± 6)	14	(13 ± 1)
		83		6		41		38		14
		83		3		38		28		12
	1250*	92	(79 ± 14)	4	(9 ± 5)	32	(34 ± 3)	24	(26 ± 3)	13	(10 ± 3)
		64		14		34		25		7
		80		8		37		30		9
	2500*	60	(76 ± 15)	4	(7 ± 3)	39	(44 ± 5)	15	(20 ± 5)	9	(10 ± 2)
		81		9		48		20		9
		88		9		44		24		13
	5000*	69	(69 ± 4)	5	(7 ± 3)	36	(32 ± 4)	28	(23 ± 4)	8	(9 ± 3)
		73		7		20		22		13
		65		10		31		20		7
Positive control -S9 mix	Chemical	AF-2		NaN3		AF-2		AF-2		ICR-191
	Dose ((µg/plate)	0.01		0.5		0.01		0.1		0.5
	No. of revertant colonies/plate	943	(966 ± 57)	340	(353 ± 19)	141	(125 ± 14)	420	(416 ± 11)	442	(458 ± 16)
		1031		344		121		424		473
		925		374		113		404		459
Positive control +S9 mix	Chemical	2AA		2AA		2AA		B[a]P		2AA
	Dose ((µg/plate)	1		2		10		5		2
	No. of revertant colonies/plate	1230	(1238 ± 14)	210	(187 ± 36)	574	(557 ± 31)	288	(270 ± 26)	162	(157 ± 9)
		1255		146		521		281		147
		1230		206		575		240		162

Note: ( ) The mean number of revertant colonies per plate ± standard deviation (n=3)

* Precipitation of the test item was observed in all plates for each tester strain

AF-2: furylfuramide

NaN3: Sodium azide

2AA: 2-aminoanthracene

B[a]P: Benzo[a]pyrene 

 

Table 4: Historical data (Main test 1) Negative control (Mean+/-3 S.D.)

	-S9 mix					+S9 mix
	TA100	TA1535	WP2uvrA	TA98	TA1537	TA100	TA1535	WP2uvrA	TA98	TA1537
Mean	118	10	33	20	11	109	10	29	27	12
S.D.	18	4	12	6	4	17	3	11	7	4
Upper limit	172	22	69	38	23	160	19	62	48	24
Lower limit	64	1	1	2	1	58	1	1	6	1

Table 5: Historical data (Main test 1) Positive control (Mean+/-3 S.D.)

	-S9 mix					+S9 mix
	TA100	TA1535	WP2uvrA	TA98	TA1537	TA100	TA1535	WP2uvrA	TA98	TA1537
Chemical	AF-2	NaN3	AF-2	AF-2	ICR-191	2AA	2AA	2AA	B[a]P	2AA
Dose (µg/plate)	0.01	0.5	0.01	0.1	0.5	1	2	10	5	2
Mean	869	282	145	463	604	1476	237	458	264	205
S.D.	89	47	29	56	95	261	35	121	36	38
Upper limit	1136	423	232	631	889	2259	342	821	372	319
Lower limit	602	141	58	295	319	693	132	95	156	91

Test period from January – June 2018, negative control and positive control with S9 mix

Test period from February – June 2018, positive control without S9 mix

 

Table 6: Historical data (Main test 2) Negative control (Mean+/-3 S.D.)

	-S9 mix					+S9 mix
	TA100	TA1535	WP2uvrA	TA98	TA1537	TA100	TA1535	WP2uvrA	TA98	TA1537
Mean	115	10	31	20	11	107	10	28	27	12
S.D.	18	3	9	6	5	17	3	9	8	8
Upper limit	169	19	58	38	26	158	19	55	51	27
Lower limit	61	1	4	2	1	56	1	1	3	1

Table 7: Historical data (Main test 2) Positive control (Mean+/-3 S.D.)

	-S9 mix					+S9 mix
	TA100	TA1535	WP2uvrA	TA98	TA1537	TA100	TA1535	WP2uvrA	TA98	TA1537
Chemical	AF-2	NaN3	AF-2	AF-2	ICR-191	2AA	2AA	2AA	B[a]P	2AA
Dose (µg/plate)	00.1	0.5	0.01	0.1	0.5	1	2	10	5	2
Mean	856	289	141	463	599	1421	232	452	263	198
S.D.	99	54	29	54	103	308	39	120	37	41
Upper limit	1153	451	228	625	908	2345	349	812	374	321
Lower limit	559	127	54	301	290	497	115	92	152	75

Test period from February 2018 – July 2018

Applicant's summary and conclusion

Conclusions:

The test item had no ability to induce mutations under the test conditions.